JP2002521065A - HERG-mutation in the long-term QT syndrome gene and its genomic structure - Google Patents

Abstract

  (57) [Summary] The present invention relates to the determination of the genomic structure of HERG, a gene associated with long-term QT syndrome. The sequence of the 15 intron / exon junctions has been determined and this information is useful for designing primers for amplification and for sequencing across all exons of the gene. This is useful for determining the presence or absence of a mutation known to cause long-term QT syndrome. Also disclosed are a number of new mutations in HERG that have been found to be associated with long-term QT syndrome.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application was made with government support under grant number P50-HL52338-02. The United States government may have certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application Ser. It is.

BACKGROUND OF THE INVENTION [0002] The present invention is directed to a method for diagnosing long-term QT syndrome (LQT). LQT is HE
It has been associated with specific genes including RG, SCN5A, KVLQT1 and KCNE1. LQT is hereditary and may be due to a particular mutation in the gene, or it may be due to treatment with an agent administered to treat cardiac arrhythmia, or an antibiotic such as an antihistamine or erythromycin, for example. It can be obtained as a result of treatment with other types of drug therapy. The acquired form of LQT is the more prevalent form of the disease. It has previously been shown that the HERG gene encodes a K ⁺ channel that is involved in the acquired form of LQT. It has been shown that increasing K ⁺ levels in patients who have taken drugs to prevent cardiac arrhythmias may reduce the chances of the acquired form of LQT becoming sick and use it as a prophylactic measure.
Also, this knowledge can now be used to activate this K ⁺ channel and develop drugs that can be administered in conjunction with drugs currently used to treat cardiac arrhythmias.
Activation of K ⁺ channels must reduce the risk of developing LQT and Torsades de Points.

[0003] The publications and other materials used herein set forth the background of the invention, provide further details on the practice, are incorporated by reference, are incorporated herein by reference, Each is grouped into the attached reference list.

[0004] Sudden death due to cardiac arrhythmias is estimated to account for 11% of all natural deaths, but the mechanisms underlying arrhythmias are poorly understood (Kannel, 1987; Willic).
h et al., 1987). One form of long-term QT syndrome (LQT) is hereditary cardiac arrhythmias that cause frequent ventricular arrhythmias, especially torsades de pointes and ventricular fibrillation, causing sudden loss of consciousness, syncope and seizures (Ward, 1964; Romano, 1965; Schwartz et al.,
1975; Moss et al., 1991). The disease usually occurs in young and healthy individuals (
Ward, 1964; Romano, 1965; Schwartz, 1975). Most LQT genes carry an apparent prolongation of the QT interval in electrocardiograms, a sign of abnormal cardiac repolarization (Vinc
ent et al., 1992). The clinical features of LQT arise from episode cardiac arrhythmias, in particular the Torsades de Points, named for the characteristic waveform properties of the electrocardiogram in this arrhythmia. Torsades de Pointes can degenerate to ventricular fibrillation, especially lethal arrhythmias. LQT is not a common diagnosis, but ventricular arrhythmias are very common; 300,000 US citizens die suddenly each year (Kannel et al., 198).
7; Willich et al., 1987). And, in many cases, the underlying mechanism can be abnormal cardiac repolarization. Thus, LQT offers a unique opportunity to study life-threatening cardiac arrhythmias at the molecular level. The more common form of the disease is called "acquired LQT" and can be induced by a number of different factors, especially certain medications and treatments with reduced serum K ⁺ levels (hypocalcemia).

[0005] Autosomal dominant and autosomal recessive forms of the disease have been reported.
Autosomal recessive LQT (also known as Jervil-Lang-Nielsen syndrome) has been associated with congenital neurological deafness; this form of LQT is rare (Jervell and Lange-Nielsen, 1957). Autosomal dominant LQT (Romano-ward syndrome) is more common and has not been associated with other phenotypic abnormalities. Diseases that closely resemble hereditary LQT can also be acquired, usually as a result of pharmacological therapy (Schwar
tz et al., 1975; Zipes, 1987).

In 1991, a complete linkage between autosomal dominant LQT and polymorphism in HARS was reported (Keating et al., 1991a; Keating et al., 1991b). This finding maps LQT1 to chromosome 11p15.5, allowing presymptomatic diagnosis in some families. Autosomal dominant LQT was previously thought to be genetically homogeneous and the first 7 families studied were linked to 11p15.5 (Kea
ting et al., 1991b). In 1993, it was found that there was a locus heterogeneity in LQT (Benhorin et al., 1993; Curran et al., 1993b; Towbin et al., 1994). Then 2
Additional LQT loci: LQT2 on chromosome 7q35-36 (9 families) and LQT3 on 3p21-24 (3 families) were identified (Jiang et al., 1994). Genes that contribute to LQT at these loci were subsequently identified. These are KVLQT1 (LQT1), HERG (LQT2) and SCN5A (Wang et al., 1996; Curran et al., 1995; Wang et al., 1995; US Pat. No. 5,599,673). Later, KCNE1 (LQT5) was also associated with long-term QT syndrome (Splawski et al.,
1997; Duggal et al., 1998). These genes encode ion channels involved in the generation of cardiac action potentials. Mutations can lead to loss of channel function and delayed cardiac cell repolarization. Abnormal cardiac repolarization creates the basis for arrhythmias due to regional heterogeneity of channel expression in the myocardium. KVLQT1 and KCNE1 are also expressed in the inner ear (Neyroud et al., 1997; Vetter et al., 1996). It has been shown that homozygous or compound heterozygous mutations in these genes can cause severe phenotypes of deafness and Jaber-Lange-Nielsen syndrome (Neyrou
d et al., 1997; Splawski et al., 1997; Schultze-Bahr et al., 1997; Tyson et al., 1997). Loss of functional channels in the ear apparently destroys the production of endolymph and leads to deafness. In some families, it remains unlinked to a known locus,
This suggests additional locus heterogeneity for LQT. This degree of heterogeneity suggests that different LQT genes may interact to encode proteins that regulate cardiac repolarization and arrhythmia risk.

[0007] Currently, presymptomatic diagnosis of LQT is based on the delay of the QT interval on an electrocardiogram. 0.
A QTc (QT interval corrected for heart rate) longer than 44 seconds traditionally classifies an individual as suffering. However, most LQT patients are young without an electrocardiogram or otherwise healthy individuals. Furthermore, genetic studies have shown that QTc is neither sensitive nor specific (Vincent et al., 1992).
. The spectra of QTc intervals for gene carriers and non-carriers overlap, leading to misclassification. Non-carriers can have a long QT interval and are diagnosed as suffering. Conversely, some LQT gene carriers have <0.44 seconds of QT
It has a c-interval but is still at high risk for arrhythmias. Accurate presymptomatic diagnosis is important for an effective gene-specific treatment of LQT.

[0008] Genetic screening using mutation analysis can improve presymptomatic diagnosis. The presence of the mutation will definitively distinguish affected individuals and will identify the genes underlying LQT, even in small families and sporadic cases. To facilitate the identification of LQT-related mutations, we determined the genomic structure of HERG and designed primer pairs to propagate each exon. Additional mutations in HERG were identified by single-stranded DNA conformation polymorphism (SSCP) analysis.

In 1994, Warmke and Ganetzky introduced a novel human cDNA, human ether
An a-go-go related gene was identified (HERG, Warmke and Ganetzky, 1994).
HERG was localized to human chromosome 7 by PCR analysis of a somatic cell hybrid panel (Warmke and Ganetzky, 1994). The function of the protein encoded by HERG is unknown, but it has deduced amino acid sequence homology to potassium channels. HERG was isolated from a hippocampus cDNA library by homology to the Drosophila ether a-go-go gene (eag) encoding a calcium-regulated potassium channel (B
ruggemann et al., 1993). HERG is not a human homolog of eag, but shares only 〜50% amino acid sequence homology. Although the function of HERG is unknown, it is strongly expressed in the heart and plays an important role in repolarizing the cardiac action potential,
It was postulated to be linked to LQT (Curran et al., 1995).

[0010] The acquired LQT is usually the heart K⁺Treatment with channel-blocking drug therapy?
(Roden, 1988). The pharmacotherapy most commonly associated with LQT is
Cardiac early activation-delayed rectification K as part of the spectrum of physical activity⁺Current, I _Kr Antiarrhythmic drugs (eg, quinidine, sotalol). Other drugs
An acquisition LQT may be triggered. These include antihistamines and erythromycin
Some antibiotics are included. I_KrIs characteristic of isolated cardiomyocytes.
(Balser et al., 1990; Follmer et al., 1992; Sanguinetti and Jurk)
iewicz, 1990; Shibasaki, 1987; T. Yang et al., 1994), initiates repolarization of action potential
It is known to have an important role in doing so.

To clarify the physiological role of HERG, full-length cDNA was cloned and the channel was expressed in Xenopus oocytes. Voltage clamp analysis of the current generated revealed that HERG encodes a K ⁺ channel with nearly the same biophysical characteristics as I _Kr . These data suggests that the HERG encodes the major subunit of the I _Kr channel, some form of genetic and drug - are subjected to machine work linkage between the induction LQT.

SUMMARY OF THE INVENTION [0012] The HERG genomic structure has been determined and shows that it contains 15 exons and extends over 55 kilobases. Primer pairs are shown to allow analysis of all 15 exons for mutations that may be associated with long-term QT syndrome.
Many new mutations in HERG associated with long-term QT syndrome are also shown.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1D: Currents induced by depolarizing potential differences in Xenopus oocytes injected with HERG cRNA. Fig. 1A: -50 to 1
Current activated by a 4 second pulse applied in 10 mV increments to 0 mV. The current during the pulse gradually increased with the voltage, like the tail current when returning to the holding potential. The holding potential was -70 mV. Inset shows voltage pulse
Figure 2 illustrates the protocol. FIG. 1B: Current activated by a test pulse from 0 to +40 mV, applied in 10 mV increments. The magnitude of the current during the pulse gradually decreased with voltage, while the tail current saturated at +10 mV. Note that the current did not show slow inactivation. FIG. 1C: Current-voltage relationship to peak HERG current recorded during a 4 second pulse (n = 10). FIG. 1D: HE
Voltage-dependence of RG channel activation. Tail current amplification was measured at -70 mV after a 4 second pulse and then normalized to maximum current. Data are Bolzmann coefficients:
I = 1 / (1 + exp [(Vt-V 1/2) k]), where, I = relative tail current, _{V t} = test _{potential, V 1/2} is the voltage required for half activation current And k is the slope coefficient (V _1/2 = −15.1 ± 0.6 mV; k = 7.85 ± 0.2 mV, n =
10).

FIG. 2A-D: Dynamics of HERG current activation and deactivation FIG. 2A: Activation current is activated by a 3.25 second pulse to a test potential ranked from −50 to +20 mV (10 mV step). did. The current and corresponding single exponential fit (I = A ₀ + A ₁ e ^{−t / t} ) are overlaid. FIG. 2B: Deactivation of HERG current. The current was activated by a 1.6 second pulse to +20 mV, followed by a return to the test potential, which was ranked from -40 to -100 mV (10 mV steps).
Deactivation current and corresponding double exponential fit (I _tail = A ₀ + AMP _f · ex
It is repeated _{^{p -t / tf + AMP s ·}} exp-t / ts). The current was not leaky. FIG. 2C: Activation (n = 15) and rapid deactivation (n =
11) Voltage-dependent dynamics. FIG. 2D: H as a function of test potential (n = 11)
Time constants (t _f , t _s ) and relative amplification of the early (AMP _f ) and late (AMP _s ) components of ERG current deactivation. Relative amplification was not measured at -80 and position 90 mV due to the small current magnitude near the reversal potential.

FIG. 3A-C: The reverse potential of the HERG current is K⁺-Anticipate about selectivity channels
[K⁺]_eHas changed. FIG. 3A: Tail current pulsed to +20 mV
-105 to -80 mV in oocytes soaked in ND96 solution after
E) (applied in 5 mV steps). Approximate reversal potential of tail current is -97 mV
Met. The current was not leaky. Figure 3B: tail current modified
ND96 solution ([K⁺]_e= 75 mM in the same oocyte soaked in 10 mM)
At a potential of -50 mV (applied in 5 mV steps). Reversal potential of tail current
Was -65 mV. FIG. 3C: Inversion potential of HERG current (E_rev) Is [K⁺] _e Varies as a function of E_revIs the tail current amplification as a function of the test potential.
Measure for each oocyte by determining zero-intercept from the plot.
Specified. The data is 2 mM [K⁺]_eExcept for (n = 15), the average of the 5 measurements
Show. The dotted line is given by the Nernst equation for the complete K + -selective channel.
This is the expected relationship. The solid curve is the Goldman-Hodgkin-Katz current equation (
Goldman, 1943; Hodgkin and Katz, 1949): E_rev= 58 log ｛(r [Na⁺] _e + [K⁺]_e) / (R [Na⁺]_i+ [K⁺]_i) Shows the conformity of the data to ①. This
K determined from the fit of⁺Na for⁺Has a relative transmittance (r) of 0.007.
Was.

FIGS. 4A-E: Extracellular K⁺Activation of HERG current. Figures 4A-C: 10 mM
 Eggs immersed in a modified ND96 solution containing KCl (A) and 2 mM KCl (B)
Replace after 5 minutes with ND96 solution in mother cells or without KCl
4 seconds pulse to test potential of rank -50 to +20 mV in obtained oocytes
Current induced by FIG. 4D: Current-voltage versus current shown in panels AC
Relationship. Figure 4E: HERG current amplification is [K⁺]_eVaries as a function of The current is
It was measured at a test potential of +20 mV (n = 4-6). Solid line is straight line fit to data
(I_HERG= 189 + 37.5 · [K⁺]_e). Low and high [K⁺] _e This relationship in [K⁺]_eWhat would not be expected to be a linear relationship of
Please note.

FIGS. 5A-D: HERG rectification resulting from rapid inactivation. FIG. 5A: +20, 0, after activation with a 260 ms pulse to +40 mV ([K ⁺ ] _e = 10 mM).
Currents recorded at test potentials of -40 and -70 to -120 mV (in 10 mV steps). The current was recorded at a sampling rate of 10 kHz. Only the 90 ms tail current following the last 30 ms of the activation pulse is shown. Leakage current was removed using P / 3 subtraction; the first 2 ms of tail current was left. The tail currents recorded at several potentials (+20 to -60 mV) were fit with a single exponential, since the deactivation was slow enough that it did not significantly contribute to the tail current net kinetics. At more negative potentials (-70 to -120 mV), the currents matched the dual function calculated for the early phase of deactivation, which overlapped with recovery from inactivation. The fit to the data is overlaid on the current trajectory. FIG. 5B: Time constant for recovery from rapid inactivation determined from tail current adaptation described above. FIG. 5C: Fully activated HER
GIV relationship. The maximum conductivity (118 μS) of the HERG current is −90 and −12.
It was determined from the slope of the first order fit to the current amplitude at a potential between 0 mV. FIG. 5D
: Voltage dependence of rapid inactivation of HERG current. Rectification coefficient at each potential (rectif
ication factor), R was calculated using the current amplitude plotted in panel (C):

R = [G · n · (V _t −E _rev )] / I _HERG

Where G = maximum conductivity of HERG (118 μS); activation variable at n = + 40 mV (1.0); V _t = test potential; E _rev = reversal potential (−73 mV). The data was fit with the Boltzmann equation: 1 / (1 + exp [(E _rev −V _1/2 ) / k]). The value of V1 _{/ 2} was -49 mV, and the slope coefficient (k) was +28 mV.

FIG. 6A-D: HERG current is interrupted by La ³⁺ . FIG. 6A: Control current activated by a 4 second pulse from -50 to a potential ranked +50 mV. The current did not leak. FIG. 6B: Currents evoked by the same pulse protocol after exposing oocytes to 10 μM LaCl ₃ . FIG. 6C: IV relationship of HERG current measured at the end of the 4 second test pulse. FIG. 6D: Isochronal activation curves were determined from plots of tail current amplitude as a function of test potential. The data was fitted to a Boltzmann function to obtain a smooth isochronal activation curve. La ³⁺ shifted the half-point of activation from -16 mV to +23 mV.

FIG. 7: HERG physical map and exon configuration. The genomic region of HERG encompasses approximately 55 kilobases. 2 shows duplicate cosmid clones containing the entire HERG transcribed sequence. The location of the HERG exon relative to the genomic clone is shown.
Exon sizes and distances are not drawn to scale.

FIG. 8A-B: Genomic set organization of HERG coding sequence and 5 ′ and 3 ′ untranslated sequences. The position of the intron is indicated by an arrow. 6 estimated membrane spanning segments (
The spanning segment (S1 to S6S) and the putative pore (Pore) and cyclic nucleotide binding (cNBD) regions are underlined. The asterisk mark is a stop codon. The nucleic acids and proteins of FIGS. 8A-B are, respectively, SEQ ID NO: 3.
And SEQ ID NO: 4.

9A-E: Pedigree structure and genotype analysis of 5 new LQT families. Individuals exhibiting characteristic features of LQT, including delayed QT intervals and a history of syncope, seizures or sudden stunted death are indicated by solid circles (females) or squares (males). Unaffected individuals are indicated by open circles or squares. Individuals with an ambiguous phenotype or for whom phenotypic data was not available are stippled. Shaded circles or squares indicate individuals who died. Haplotypes for polymorphic markers linked to LQT2 are shown below each individual. These markers include (from centromere to telomere) D7S505, D7S636, HERG5-11, HERG3-8
, D7S483 (Gyapay et al., 1994; Wang et al., 1995). Haplotypes that cosegregate with the disease phenotype are indicated by boxes. Recombination events are indicated by horizontal black lines. Informed consent was obtained from all individuals or their parents according to the Local Institutional Review Board Guidelines. Haplotype analysis indicates that the LQT phenotype in these relatives is chromosome 7q35-36.
It shows that it is linked to the above marker.

FIGS. 10A-C: HEQ gene deletion associated with LQT in 2 families. K
Pedigree structure of 2287 (FIG. 10A), results of PCR amplification using primer pair 1-9 (FIG. 10A), DNA of normal and mutant L2278HERG gene
The results of sequencing (FIG. 10B), as well as the effect of a putative structural deletion of the HERG protein (FIG. 10C) are shown. It is noted that an abnormal 143 bp fragment was observed in affected members of this kin, indicating the presence of a disease-associated intragenic deletion. The DNA sequence of the normal and abnormal PCR products had a 27 bp deletion (
ΔI500-F508). This mutation causes a third membrane spanning
Causes an in-frame deletion of 9 amino acids in domain (S3). Shows the deleted sequence.

11A-C: Shows the K2595 family tree structure (FIG. 11A). Individuals who died are indicated by diagonal lines. The results of SSCP analysis using primer pair 1-9 are shown below each individual (FIG. 11A). It is noted that abnormal SSCP conformers co-segregate with disease in this family. The DNA sequence shows a single base pair deletion ($ 1261) (FIG. 11B). This deletion creates a stop codon 12 amino acids downstream following the frameshift (FIG. 11C). Deleted nucleotides are indicated by arrows.

FIG. 12A-I: HERG point mutations were identified in 3 LQT relatives.
K1956 (FIG. 12A), K2596 (FIG. 12C) and K2015 (FIG. 12E).
2) shows the family tree structure. The primer pair 5-11 (K19
56) (FIG. 12B), the results of SSCP analysis using primer pair 1-9 (K2596) (FIG. 12D) and primer pair 4-12 (K2015) (FIG. 12F). Aberrant SSCP conformers co-segregate with disease in each kin. DNA sequence analysis of normal and abnormal conformers revealed K195
The C to T substitution at position 1682 in 6 was revealed. This mutation results in a substitution of valine for a highly conserved alanine residue at codon 561 (A561V) (FIG. 12G). Analysis of K2596 revealed an A to G substitution at position 1408 (T on the antisense strand).
(Showing substitution of C to C) (FIG. 12D). This mutation results in a substitution of aspartic acid for a conserved asparagine in the second membrane spanning domain (
N470D) (Figure 12H). Analysis of K2015 revealed a G to C substitution (indicating a C to G substitution on the antisense strand) (Figure 12F). This mutation occurs in the splice-donor sequence of intron III (Curran
Et al., 1995) (Intron 9 herein) (Figure 121). The coding sequence is the upper case and the intron sequence is the lower case. G to C
Note that the substitution to destroys the splice-donor site. (HERG, M
-Eag, elk, Warmke and Ganetzky, 1994; R-eag; Ludwig et al., 1994).

FIGS. 13A-E: HERG missense mutations associated with LQT. The results from the SSCP analysis and the effect of the mutation on the amino acid sequence are shown below each pedigree.
Note that the abnormal SSCP conformer (indicated by the arrow) is cosegregated with the disease phenotype. Figures 14A-C: HERG de novo mutations in sporadic cases of LQT. K
2269 pedigree structure (FIG. 14A) and SSCP analysis (primer pair 14
-16) (Figure 14A) shows an abnormal conformer in sporadic cases of LQT. DNA sequence analysis identified a G to A substitution at position 1882 of the cDNA sequence (indicating a C to T substitution on the antisense strand) (FIG. 14B). Note that this mutation results in a serine substitution (G628S) for a highly conserved glycine residue at codon 628 (FIG. 14).
C). This amino acid sequence is known to be important for potassium ion selectivity.

FIG. 15: Northern blot analysis of HERG mRNA showing strong expression in heart. Northern blots (Clonetech, poly A ⁺ RNA, 2 mg / lane) were probed with HERG cDNA containing nucleotides 679 to 2239 of the coding sequence. Two cardiac mRNAs of -4.1 and 4.4 kb were shown.
Although the background of mRNA extracted from the lung was high, no specific band was identified.

Outline of Sequence Listing SEQ ID NO: 1 is only the nucleic acid coding region of HERG cDNA. SEQ ID NO: 2 is the HERG protein encoded by SEQ ID NO: 1. SEQ ID NO: 3 is the nucleic acid of the HERG cDNA, including the complete coding region and some 5 'and 3' untranslated regions. SEQ ID NO: 4 is a HERG protein encoded by SEQ ID NO: 3. SEQ ID NOs: 5 and 6 are hypothetical nucleic acids used to demonstrate calculated% homology. SEQ ID NOs: 7 and 8 are primers for amplifying the HERG 3′UTR. SEQ ID NOs: 9-25 are primer pairs for SSCP analysis (Table 3). SEQ ID NOs: 26-55 are the intron / exon boundaries of HERG (Table 4)
). SEQ ID NOs: 56-95 are primers for amplifying the HERG exon (Table 5). SEQ ID NOs: 96-97 show the deletion of K2287 (FIG. 10C). SEQ ID NOs: 98-101 show the effect of the deletion at K2595 (FIG. 11C
). SEQ ID NOs: 102-116 are comparisons of regions of HERG from human, mouse, rat and Drosophila (FIGS. 12G-H and 14C).

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the genomic structure of HERG, as well as to newly discovered mutations in HERG associated with LQT. In addition, the present invention provides a HER that causes LQT.
It is directed to a method of screening a human for the presence of a G gene variant.
Because LQT can now be detected earlier (ie, before symptoms appear) and more clearly, better treatment options will be available in individuals identified as having hereditary LQT. The present invention provides a method for screening the HERG gene to identify mutations. Further, such methods can include amplifying a portion of the HERG gene, and further include providing a series of polynucleotides that are primers for amplification of the portion of the HERG gene. This method is useful for identifying mutations for use in either diagnosing or predicting LQT.

[0030] Long-term QT syndrome is a hereditary or acquired disease that causes sudden death from cardiac arrhythmias, specifically Torsades de Pointes and ventricular fibrillation. LQT has previously been identified at 4 loci: KVLQT1 on chromosome 11p15.5, HERG on 7q35-36, SCN5A on 3p21-24 and chromosome 21q22.1-22.
2 mapped to KCNE1.

Evidence that the HERG gene is involved in causing hereditary LQT is demonstrated by finding sequences in DNA extracted from affected HERG gene products or affected relatives that produce abnormal levels of gene products. can get. Such LQT-sensitive alleles will co-segregate with disease in large relatives. They will also be much more frequent in unrelated individuals with LQT than in individuals in the general population. The key is to find mutations that are significant enough to cause obvious disruption to the normal function of the gene product. These mutations can take many forms. The most severe forms will be frameshift mutations or large deletions that will result in genes encoding abnormal proteins or will significantly alter protein expression. Less severe disruptive mutations include changes in basic amino acids to or from cysteine residues, or vice versa, or hydrophobic amino acids to hydrophilic amino acids or vice versa. Includes small in-frame deletions and non-conservative base pair substitutions that would have a significant effect on the protein produced, or other mutations that would affect secondary or tertiary protein structure Will be. Those that produce silent mutations or conservative amino acid substitutions will generally not be expected to disrupt protein function.

According to the diagnosis and prediction method of the present invention, a change in the wild-type HERG gene is detected. In addition, the method can be performed by detecting the wild-type HERG gene and confirming the lack of LQT development as a result of a mutation at this locus. "Wild-type gene alterations" include all forms of mutations, including deletions, insertions and point mutations in coding and non-coding regions. Deletions can be of the entire gene or only a portion of the gene. Point mutations can result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those that occur only in certain tissues and are not inherited in the germline. Germline mutations can be found in any of the tissues of the body and are inherited. The point mutation event is m
It can occur in a regulatory region, such as in the promoter of a gene, which leads to a loss or reduction in RNA expression. Point mutations can result in loss of expression of the HERG gene product or m
Appropriate RNA processing, which leads to a decrease in RNA stability or translation efficiency, may also be abolished.

The presence of hereditary LQT can be confirmed by testing any human tissue for mutations in the HERG gene. For example, individuals who have inherited a germline HERG mutation will be more likely to develop LQT. This can be determined by testing DNA from any tissue of the individual's body. Most simply, blood can be collected and DNA can be extracted from cells of the blood. In addition, prenatal diagnosis is based on HE
This can be done by testing fetal, placental or amniotic cells for mutations in the RG gene. An alteration in a wild-type HERG allele, whether due to a point mutation or a deletion, for example, can be detected by any of the methods discussed herein.

There are several methods that can be used to detect DNA sequence variations. Sequence variation can be detected by direct DNA sequencing, either manual sequencing or automated fluorescent sequencing. Another approach is the single-stranded DNA conformation polymorphism assay (SSCP) (Orita et al., 1989). This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variations. While reduced detection sensitivity is disadvantageous, the increased throughput possible with SSCP makes direct sequencing an attractive, valuable alternative for research-based mutation detection. The fragments with shifted mobility on the SSCP gel are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on detecting mismatches between two complementary DNA strands include clamped denaturing gel electrophoresis.
ring gel electrophoresis) (CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White et al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989) ) Is included. None of the methods described above will detect large deletions, duplications or insertions, or they will not detect regulatory mutations that affect protein transcription or translation. Other methods that could detect these classes of mutations, such as protein truncation assays or asymmetric assays, would detect only certain types of mutations and not missense mutations. A review of currently available methods for detecting DNA sequence variations can be found in a recent review by Grompe (1993). Once the mutation is known, the allele-specific oligonucleotide (AS
O) An allele-specific detection approach, such as hybridization, can be used to rapidly screen many other samples for the same mutation. Such techniques can utilize probes labeled with gold nanoparticles to obtain visible color results (Elghanian et al., 1997).

A rapid preliminary analysis to detect polymorphisms in DNA sequences is to generate a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with multiple restriction enzymes. This can be done by considering. Each blot contains a series of normal individuals and a series of LQT cases. Southern blots showing hybridizing fragments (different in length than control DNA when probed with sequences near or containing the HERG locus) indicate possible mutations. If restriction enzymes that produce very large restriction fragments are used, then pulse field gel electrophoresis (PFGE) is used.

Detection of point mutations can be performed by molecularly cloning the HERG allele and sequencing the allele using techniques well known in the art. Also, the gene or portion of the gene can be amplified, for example, by PCR or other amplification techniques, and the amplified gene or portion of the amplified gene can be sequenced. There are six well-known methods for more complete but still indirect testing to confirm the presence of susceptibility alleles: 1) Single-stranded DNA conformation analysis (
SSCP) (Orita et al., 1989); 2) Denaturing gradient gel electrophoresis (DGGE
) (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection assay (Fi
nkelstein et al., 1990; Kinszler et al., 1991); 4) Allele-specific oligonucleotides (ASOs) (Conner et al., 1983); 5) E. coli mutS.
Use of proteins that recognize nucleotide mismatches, such as proteins (Modric
h, 1991); and 6) Allele-specific PCR (Ruano and Kidd, 1989).
For allele-specific PCR, a primer is used that hybridizes at the 3 'end to a particular HERG mutation. If no particular mutation is present, no amplification product is observed. As disclosed in European Patent Application Publication No. 0332435 and Newton et al., 1989, the Amplification Refracto
ry Mutation System) (ARMS) can also be used. Gene insertions and deletions can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to assess changes in alleles or insertions in polymorphic fragments. Such methods are particularly useful for screening relatives of an affected individual for the presence of the mutation found in that individual. Other techniques for detecting insertions and deletions known in the art may be used.

In the first three methods (SSCP, DGGE and RNase protection assays), new electrophoretic bands appear. SSCP detects bands that migrate differently because sequence changes cause differences in single strands, intramolecular base pairing. R
Nase protection involves cleavage of the mutated polynucleotide into two or more fragments. DGGE uses a denaturing gradient gel to detect differences in the migration rate of the mutated sequence compared to the wild-type sequence. Allele-
In specific oligonucleotide assays, oligonucleotides that detect specific sequences are designed, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain nucleotide mismatches in the heteroduplex between the mutant and wild-type sequences.

According to the present invention, a mismatch is a hybridized nucleic acid duplex in which the two strands are not 100% complementary. The lack of overall homology may be due to a deletion, insertion, inversion or substitution. Mismatch detection can be used to detect point mutations in a gene or its mRNA product. Although these techniques are less sensitive than sequencing, they are simpler to perform on large numbers of samples. One example of a mismatched cutting technology is RN
It is the case protection law. In the practice of the present invention, the method involves the use of a labeled riboprobe that is complementary to the human wild-type HERG gene coding sequence. The riboprobe is annealed together with either mRNA or DNA isolated from the individual, and then digested with the enzyme RNase A, which can detect some mismatches in the duplex RNA structure. If a mismatch is detected by RNase A, it cuts the site of the mismatch. Mismatches are then detected when the annealed RNA preparation is separated on an electrophoresis gel matrix, and when cleaved by RNase A, the riboprobe and mRNA or DN.
An RNA product smaller than the full-length duplex RNA for A will be seen. The riboprobe need not be the full length of the mRNA or gene, but can be any segment. If the riboprobe contains only mRNA or a segment of the gene, a large number of these probes may be used to match the entire mRN for mismatch.
It may be desirable to screen for the A sequence.

In a similar manner, mismatches can be detected with a DNA probe via enzymatic or chemical cleavage. See, for example, Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches may be detected by a shift in the electrophoretic mobility of the mismatched duplex relative to the matched duplex. For example, Ca
See riello, 1988. Using either riboprobes or DNA probes, cellular mRNA or DNA that may contain mutations can be amplified using PCR before hybridization (see below). Changes in the DNA of the HERG gene can also be detected using Southern hybridization, especially if the changes are large rearrangements such as deletions and insertions.

The DNA sequence of the HERG gene amplified by using PCR can also be screened using an allele-specific probe. These probes are nucleic acid oligomers, each containing a region of the gene sequence containing a known mutation. For example, one oligomer has about 30 corresponding to a portion of the gene sequence.
It can be nucleotide length. By using a series of such allele-specific probes, PCR amplification by-products can be screened to identify the presence of previously identified mutations in the gene. Hybridization of the amplified HERG sequence with an allele-specific probe can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates that the same mutation is present in the tissue as in the allele-specific probe.

A technique of nucleic acid analysis newly developed through a microchip technique is also applicable to the present invention. In this technique, literally, thousands of different oligonucleotide probes are constructed in an array on a silicon chip. The nucleic acid to be analyzed is fluorescently labeled and hybridized to a probe on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips.
Using this technique, one can determine the presence of a mutation or even sequence the nucleic acid to be analyzed or measure the expression level of the gene of interest. This method is one of many, even thousands of simultaneous, parallel processing of probes, which can significantly increase analysis speed. Several papers using this technology have been published. Some of these are Hacia et al., 1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996; DeRisi et al., 1996; Lipshutz et al., 1995.
It is. This method has already been used to screen people for mutations in the breast cancer gene BRCA1 (Hacia et al., 1996). This new technology has been reviewed in a new article in Chemical and Engineering (Borman, 1996) and has been the subject of editorials (Editorial, Nature Genetics, 1996). Fo
See also dor (1997).

The most complete test for mutations in candidate loci is to directly compare genomic HERG sequences from patients to those from a control population. Alternatively,
Messenger RNA can be sequenced after amplification, for example by PCR, thereby obviating the need to determine the exon structure of a candidate gene. Mutations from patients outside the coding region of HERG can be detected by examining non-coding regions, such as introns and regulatory sequences near or in the gene. Early indications that mutations in noncoding regions are important may have come from Northern blot experiments that revealed abnormal size or abundant messenger RNA molecules in patients compared to control individuals .

[0043] Alterations in HERG mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and R
Includes Nase protection. A decrease in mRNA expression indicates a change in the wild-type gene.
Changes in the wild-type gene can also be detected by screening for changes in the wild-type HERG protein. For example, tissues can be screened using a monoclonal antibody immunoreactive with HERG. Lack of relative antigen will indicate a mutation. An antibody specific for the product of the mutant allele could be used to detect the mutant gene product. Such an immunoassay may be performed in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA assays. H changed
Any method of detecting ERG protein can be used to detect changes in the wild-type HERG gene. Functional assays such as protein binding determinations can be used.
In addition, assays that detect the biochemical function of HERG may be used. Mutant HE
Finding the RG gene product indicates a change in the wild type HERG gene.

[0044] Mutant HERG genes or gene products can also be detected in other human body samples, such as serum, stool, urine and saliva. The same techniques discussed above for detecting mutated genes or gene products in tissues can be applied to other body samples. By screening such body samples, a simple early diagnosis for hereditary LQT can be achieved.

The primer pairs of the present invention are useful for determining the nucleotide sequence of a particular HERG allele using PCR. The pair of single-stranded DNA primers
To initiate amplified DNA synthesis of the gene itself, it may be annealed to the sequence in or surrounding the HERG gene on chromosome 7. The complete set of these primers allows for the synthesis of the gene coding sequence, ie, all nucleotides of the exon. Preferably, the set of primers permits the synthesis of both intron and exon sequences. Allele specific primers may also be used. Such primers will anneal only to specific HERG mutant alleles and will therefore amplify the product only in the presence of the mutant allele as a template.

To facilitate subsequent cloning of the amplified sequence, the primer is
It may have a restriction enzyme site sequence attached to the end. Thus, all nucleotides of the primer are from the HERG sequence or a sequence adjacent to the HERG, except for a few nucleotides required to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques well known in the art. Generally, primers can be made using commercially available oligonucleotide synthesizers. HERG cDNA obtained
Sequences (Warmk and Ganetzky, 1994), the design of particular primers, are well known in the art. The present invention adds to this the ability to present data on intron / exon boundaries so that primers can be designed to completely amplify and sequence the entire exon region.

The nucleic acid probes provided by the present invention are useful for a number of purposes. that is,
It can be used in Southern hybridizations to genomic DNA and in RNase protection methods to detect point mutations already discussed above. The probe can be used to detect the PCR amplification product. They can also detect mismatches with the HERG gene or mRNA using other techniques.

It was discovered that individuals with the wild-type HERG gene did not have hereditary LQT. However, mutations that interfere with the function of the HERG gene product do not contribute to LQT pathology. Thus, loss of function or the presence of an altered (or mutated) HERG gene that produces a protein with altered function directly triggers LQT, which increases the risk of cardiac arrhythmias. To detect HERG gene mutations, a biological sample is prepared and analyzed for differences between the sequence of the allele to be analyzed and the sequence of the wild-type allele. Mutant HERG alleles can be initially identified by any of the techniques described above. The mutant allele is then sequenced to identify specific mutations of the particular mutant allele. Alternatively, mutant alleles can be identified initially by identifying the mutated (altered) protein using conventional techniques. The mutant alleles are then sequenced to identify specific mutations for each allele. Then the mutation,
In particular, those that lead to altered function proteins are used in the diagnostic and predictive methods of the invention. The present invention also provides a method of treating a patient with K ⁺ to reduce the chance of developing LQT and / or Torsades de Points. Modulation of HERG by extracellular K ⁺ ([K +] _e ) may have physiological significance. Rapid heart rate
te) or during ischemia, K ⁺ accumulates in intracellular fissures (Gintant et al., 1992).
). This increase in [K ⁺ ] _e will increase the contribution of HERG to the net repolarization current. Thus, HERG can be even more important in modulating action potentials at high cardiac velocities or during the early phase of ischemia.

Definitions The present invention uses the following definitions: “Polynucleotide amplification” refers to polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Use the method you like. In addition, strand displacement amplification (SDA), thermophilic S
DA, and nucleic acid sequence based amplification (3SR or NASBA) are also useful. These methods are well known in the art and are widely practiced. For example,
(For PCR) U.S. Patent Nos. 4,683,195 and 4,683,202 and Inni
s et al., 1990; Wu and Wallace, 1989 (for LCR); (for SDA).
See U.S. Patent Nos. 5,270,184 and 5,455,166 and Walker et al., 1992; Spargo et al., 1996 (for thermophilic SDA) and U.S. Patent Nos. 5,409,818; Hello.
Reagents and hardware for performing PCR are commercially available. Primers useful for amplifying sequences from the HERG region are preferably complementary to sequences in the HERG region or in the region flanking the target region therein, and will specifically hybridize. The HERG sequence generated by the amplification can be sequenced directly. Alternatively, but less preferably, the amplified sequence (s) can be cloned prior to sequence analysis. Methods for direct cloning and sequence analysis of enzymatically amplified genomic segments have been described by Scharf et al., 1986.

“Analyte polynucleotide” and “analyte strand” refer to single-stranded or double-stranded polynucleotides that are predicted to contain a target sequence and are present in various types of samples, including biological samples. I can do it. “Antibodies” The present invention relates to HERG polypeptides and fragments thereof or H
Also provided are polyclonal and / or monoclonal antibodies and fragments thereof, and their immunological binding equivalents, that can specifically bind to a polynucleotide sequence from an ERG region. The term "antibody" is used to refer to either a homogeneous molecular group or a mixture, such as a serum product composed of a plurality of molecular groups. Polypeptides can be prepared synthetically on a peptide synthesizer, coupled to a carrier molecule (eg, keyhole limpet hemocyanin), and injected into rabbits over several months. Rabbit sera is tested for immunoreactivity to the HERG polypeptide or fragment. Monoclonal antibodies can be produced by injecting mice with a protein polypeptide, a fusion protein or a fragment thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with the HERG polypeptide or fragment thereof. See Harlow and Lane, 1988. These antibodies will be useful in assays as well as in medicine.

Once a sufficient amount of the desired polypeptide has been obtained, it can be used for various purposes. A typical application is the generation of antibodies specific for binding. These antibodies can be either polyclonal or monoclonal and can be produced by in vitro or in vivo techniques well known in the art. For the production of polyclonal antibodies, a suitable target immune system is selected, typically a mouse or rabbit. The substantially purified antigen is presented to the immune system in a manner determined by animal appropriate methods and other parameters well known to immunologists. Typical sites for injection are in the footpad, intramuscularly, intraperitoneally or intradermally. Of course, mice or rabbits can be replaced by other species. The polyclonal antibody, adjusted to the desired specificity, is then purified using techniques known in the art.

[0052] Immunological responses are usually assayed using an immunoassay. Usually, such immunoassays involve, for example, some purification of the origin of the antigen, which is produced by the same cells and produced in the same manner as the antigen. Various immunoassays are well known in the art. For example, Harlow and Lane, 1988 or Godi
ng, 1986.

Monoclonal antibodies having an affinity of 10 ⁻⁸ M ⁻¹ , or preferably 10 ⁻⁹ to 10 ⁻¹⁰ M ⁻¹ or more, are typically described in Harlow and Lane, 1988 or Goding, 1986. Will be generated by standard techniques that have been implemented. Briefly, the appropriate animal is selected, followed by the desired immunization protocol. After an appropriate period of time, the spleen of such an animal is removed and the individual spleen cells are typically fused to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are cloned separately and the supernatant of each clone is tested for its production of a suitable antibody specific for the region of interest of the antigen.

Other suitable techniques include in vitro lymphocyte in vitro against antigenic polypeptides (
In vitro) exposure, or alternatively, selection of a library of antibodies in phage or similar vectors. See Huse et al., 1989. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and antibodies will be labeled, covalently or non-covalently, by binding to a substance that provides a detectable signal. A wide variety of labeling and conjugate technologies are known and have been extensively reported in both the scientific and patent literature.
Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents,
Chemiluminescent agents, magnetic particles and the like are included. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350;
Nos. 4,996,345; 4,277,437; 4,275,149 and 4,366,241.
Also, recombinant immunoglobulins can be produced (see US Pat. No. 4,816,567).

“Binding partner” refers to a molecule capable of binding a ligand molecule with high specificity, such as an antigen and an antigen-specific antibody or an enzyme and an inhibitor thereof. Generally, the specific binding partner must bind with sufficient affinity to immobilize the analyte copy / (in the case of polynucleotide hybridization) complementary strand duplex under isolation conditions. Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, a vast array of known receptor-ligand conjugates and complementary polynucleotide chains. In the case of a complementary polynucleotide binding partner, the partner is usually at least about 15 bases in length and can be at least 40 bases in length. It is well recognized by those skilled in the art that lengths shorter than 15 bases (eg, 8 bases), 15 to 40 bases, and longer than 40 bases can be used. A polynucleotide may be comprised of DNA, RNA or synthetic nucleotide analogs. Additional binding partners can be identified using, for example, a two-hybrid yeast screening assay as described herein.

A “biological sample” refers to a sample of tissue or fluid that is expected to contain an analyte polynucleotide or polypeptide from an individual, including, but not limited to, plasma, serum, cerebrospinal fluid, Lymph, surface sections of skin, respiratory tract, gastrointestinal and genitourinary tracts, tears, saliva, blood cells, cancer, organs, tissues and in vitro
itro) a sample of the cell culture construct. An “encoding” polynucleotide is one that is transcribed and / or translated to produce mRNA and / or polypeptide or a fragment thereof, either in its natural state or when manipulated by methods well known to those skilled in the art. If so, it is said to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom. "Isolated" or "substantially pure""Isolated" or "substantially pure" nucleic acid (eg, RNA, DNA or mixed polymer) refers to a naturally occurring human sequence or protein, such as ribosomes, It is substantially separated from other cellular components that naturally accompany the polymerase, many other human genomic sequences and proteins. The term encompasses nucleic acid sequences or proteins removed from their naturally occurring environment and includes recombinant or cloned DNA isolates as well as chemically synthesized analogs or analogs biologically synthesized by heterogeneous systems. .

“HERG allele” refers to the normal allele of the HERG locus as well as alleles of HERG carrying mutations that cause LQT. "HERG locus", "HERG gene", "HERG nucleic acid" or "HE
"RG polynucleotide" refers to each of which certain alleles cause LQT, such that all are present in the HERG region and are expressed in normal tissues.
Refers to a polynucleotide. The HERG locus is intended to include coding sequences, intervening sequences, and regulatory elements that control transcription and / or translation. HER
The G locus is intended to include all allelic variations of the DNA sequence.

These terms, as applied to nucleic acids, refer to nucleic acids that encode polypeptides, fragments, homologs or variants of human HERG, including, for example, protein fusions or deletions. The nucleic acids of the invention can be derived from or substantially similar to a native HERG-encoding gene, or a native HERG
-Will possess sequences with substantial homology to the coding gene or a part thereof.

[0059] HERG genes or nucleic acids include silent alleles that have no effect on the amino acid sequence of a HERG polynucleotide, as well as alleles leading to amino acid sequence variants of the HERG polypeptide that do not substantially affect its function. ,
Includes normal alleles of the HERG gene. These terms also include alleles having one or more mutations that adversely affect the function of the HERG polypeptide. Mutations result in partial or complete loss of HERG function,
A change in the HERG nucleic acid sequence that produces a deleterious change in the amino acid sequence of the G polypeptide can be a change in the nucleic acid sequence that results in the loss of effective HERG expression or the production of an abnormal form of the HERG polypeptide.

The HERG nucleic acid may be as set forth in SEQ ID NO: 1 (the coding region of the HERG cDNA) or SEQ ID NO: 3 (cDNA containing the 5 ′ UTR and 3 ′ UTR), or it may be an allele as described above or the indicated sequence. Can be different from those indicated by changes that are one or more additions, insertions, deletions and substitutions of one or more nucleotides. Changes to the nucleotide sequence can result in amino acid changes at the protein level, or otherwise as determined by the genetic code.

Accordingly, the nucleic acid according to the present invention may include a sequence different from the sequences shown in SEQ ID NOs: 1 and 3, and may encode a polypeptide having the same amino acid sequence shown in SEQ ID NOs: 2 and 4. That is, the nucleic acids of the invention include sequences that are degenerate as a result of the genetic code. Alternatively, the encoded polypeptide may comprise an amino acid sequence that differs by one or more amino acid residues from the amino acid sequences shown in SEQ ID NOs: 2 and 4. Nucleic acids encoding polypeptides that are amino acid sequence variants, derivatives or alleles of the amino acid sequences shown in SEQ ID NOs: 2 and 4 are also provided by the present invention.

The HERG gene can be expressed under (a) (i) high stringency conditions (Ausubel et al., 1
992), which encodes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
Any DNA sequence that hybridizes to the complement of the NA sequence and (ii) encodes a gene product that is functionally equivalent to HERG, or (b) (i)
Hybridizing under moderately stringent conditions (Ausubel et al., 1992) to the complement of the DNA sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 under lower stringent conditions; And (ii) HERG
Also refers to any DNA sequence that encodes a gene product that is functionally equivalent to: The invention also includes nucleic acid molecules that are the complements of the sequences described herein.

The polynucleotide compositions of the present invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, as will be readily understood by those skilled in the art. Or may be biochemically modified, or may include non-natural or derivatized nucleotide bases. Such modifications include, for example,
Labeling, methylation, one or more naturally occurring substitutions with analogs, uncharged bonds (eg, methyl phosphonate, phosphotriesters, phosphoramidates, carbamates, etc.), charge bonds (eg, phosphorothioates, phosphonates, etc.) Dithiothioate, etc.), pendant groups (eg, polypeptides), intercalators (eg, acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (eg,
For example, internucleotide modifications such as alpha-anomeric nucleic acids).
Also included are synthetic molecules that mimic polynucleotides in their ability to bind to designed sequences through hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which a peptide bond is used instead of a phosphate bond in the backbone of the molecule.

The present invention provides a recombinant nucleic acid comprising all or part of a HERG region. The recombinant construct can replicate autonomously in the host cell. Alternatively, the recombinant construct may be integrated into the chromosomal DNA of the host cell. Such recombinant polynucleotides may, depending on their origin or operation, 1) not be associated with all or a portion of a polynucleotide associated therewith; 2) bind to a polynucleotide other than that which naturally binds thereto; or 3). Genome, c that does not occur in nature
Includes DNA, polynucleotides of semi-synthetic or synthetic origin. When the nucleic acid according to the present invention comprises RNA, R may be substituted for T with reference to the indicated sequence,
It must be interpreted as referring to the NA equivalent.

Thus, there is provided by the present invention a recombinant nucleic acid comprising other sequences that do not occur in nature. A wild-type sequence may also be used, but will often be altered, for example, by deletion, substitution or insertion. Various types of cDNA or genomic
Libraries can be screened as the natural source of the nucleic acids of the invention, or such nucleic acids can be sequenced from genomic DNA or other endogenous natural sources, such as PCR.
By amplification by The selection of a cDNA library usually corresponds to a tissue source rich in mRNA for the protein of interest. A phage library is usually preferred, but other types of libraries may be used. Library clones are spread out on a plate, transferred to a screening substrate, denatured, and then picked up for the presence of the sequence of interest.

[0066] DNA sequences for use in the present invention will usually include at least about 5 codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimum length required for a successful probe to specifically hybridize to the sequence encoding HERG. In this text, oligomers as short as 8 nucleotides, more usually 8-17 nucleotides, may be used for probes, especially in conjunction with chip technology.

Techniques for manipulating nucleic acids are generally described, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagents useful for applying such techniques, such as restriction enzymes, are widely known in the art and are available from New England BioLabs, Boehringer Ma.
nnheim, Amersham, Promega, USBiochemicals, New England Nuclear
It is commercially available from vendors such as Co. and many other sources. Recombinant nucleic acid sequences used to generate fusion proteins of the invention can be derived from natural or synthetic sequences. Many natural gene sequences are available from various cDNAs or from genomic libraries using appropriate probes. GenBank, National Instit
See utes of Health.

As used herein, a “portion” of a HERG locus or region or allele is at least about 8 nucleotides, or preferably about 15 nucleotides,
Or more preferably, it is defined as having a minimum size of at least about 25 nucleotides and may have a minimum size of at least about 40 nucleotides. This definition includes all sizes in the range of 8-40 nucleotides as well as those over 40 nucleotides. Therefore, this definition includes 8, 12, 15, 20, 2,
A nucleic acid of 5, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or any number of nucleotides within these values (eg, 9
, 10, 11, 16, 23, 30, 38, 50, 72, 121 other nucleotides) or nucleic acids having more than 500 nucleotides.
The present invention includes all novel nucleic acids having at least 8 nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3, its complement or a functionally equivalent nucleic acid sequence. The present invention does not include the nucleic acids present in the prior art. That is, the invention includes all nucleic acids having at least 8 nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3, but does not include nucleic acids present in the prior art.

“HERG protein” or “HERG polypeptide” refers to a protein or polypeptide encoded by a HERG locus, variant or fragment thereof. The term "polypeptide" refers to a polymer of amino acids and their equivalents and not to products of a particular length; thus, peptides, oligopeptides and proteins are included in the definition of polypeptide. included. The term does not refer to or exclude polypeptide modifications, such as, for example, glycosylation, acetylation, phosphorylation, and the like. Included within the definition are, for example, polypeptides comprising one or more amino acid analogs (including, for example, unnatural amino acids, etc.), polypeptides having substitutional bonds, and the naturally and non-naturally occurring art. Other modifications known in the art. Usually, such polypeptides are at least about 50% homologous to the native HERG sequence, preferably more than about 90% homologous, more preferably at least about 9%.
Will be 5% homologous. Also included are proteins encoded by DNA that hybridize to nucleic acids encoding HERG under high or low stringency conditions, as well as closely resembling polypeptides or proteins recovered by antisera to HERG protein (s).

The HERG polypeptide may be in SEQ ID NO: 2 or SEQ ID NO: 4, in isolated and / or purified form, which may be free or substantially free of materials with which it is naturally associated. And Polypeptides may lack natural post-translational processing, such as glycosylation, when produced by expression in prokaryotic cells or when produced synthetically. Alternatively, the invention is directed to a polypeptide that is a sequence variant, allele or derivative of a HERG polypeptide. Such polypeptides may have an amino acid sequence that differs from that set forth in SEQ ID NO: 2 or SEQ ID NO: 4 by one or more additions, substitutions, deletions or insertions of one or more amino acids. A preferred such polypeptide is H
It has an ERG function.

Substitutional variants typically involve the exchange of one amino acid for another at one or more sites in a protein without proteolytic degradation without losing other functions or properties. It may be designed to modulate one or more properties of the polypeptide, such as stability to cleavage. Amino acid substitutions may be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or thermophilic properties of the residues involved. Preferred substitutions are conservative, that is, one amino acid is replaced with one of similar form and charge. Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; Threonine; lysine, arginine; and tyrosine, phenylalanine.

Certain amino acids may have a significant loss of ability to interact with a structure, such as, for example, an antigen-binding region of an antibody or a binding site on a substrate molecule or a protein that interacts with a HERG polypeptide. Without, other amino acids can be substituted in the protein structure. Because the ability to interact with a protein determines the biological functional activity of the protein, certain amino acid substitutions can be made in the protein sequence and its underlying DNA coding sequence, and nonetheless, A protein having various characteristics can be obtained. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally understood in the art (Kyte and Doolittle, 1982).
. Alternatively, similar amino acid substitutions may be made effectively on the basis of hydrophilicity. The importance of hydrophilicity in conferring interactive biological functions of proteins is
It is generally understood in the art (US Pat. No. 4,554,101). The use of a hydrophobic index or hydrophilicity in designing polypeptides
Further discussion is provided in U.S. Patent No. 5,691,198.

The length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues. Groups, and preferably more than about 35 residues. "Operably linked" refers to a sequence that exists in a relationship that allows the components so described to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. The term peptidomimetic or mimetic is intended to refer to a substance that has the essential biological activity of a HERG polypeptide. Peptidomimetics can be peptide-containing molecules that mimic elements of protein secondary structure (Johnson et al., 1993). The underlying reason behind the use of peptidomimetics is mainly that the peptide backbone of the protein, such as that of antibodies and antigens, enzymes and substrates or scaffold proteins,
Is to direct the amino acid side chains to promote molecular interactions. Peptidomimetics are designed to allow similar molecular interactions as the natural molecule. The mimetic may not necessarily be a peptide, but it may be a natural HERG
It will retain the essential biological activity of the polypeptide.

“Probes” Polynucleotide polymorphisms associated with the HERG allele that are predisposed to LQT are likely to produce stable hybrids with polynucleotides of the target sequence under stringent to moderately stringent hybridization and wash conditions. It is detected by hybridization with the polynucleotide probe that forms. If the probe is expected to be perfectly complementary to the target sequence,
High stringency conditions will be used. Hybridization stringency may be reduced if some mismatches are expected, for example, if variants are expected with the result that the probes will not be perfectly complementary. Select conditions that eliminate non-specific / accidental binding, ie, minimize noise. (Throughout this specification, simply stating that "stringent" conditions are used means reading "using high stringency" conditions.) Since such indications identify neutral DNA polymorphisms as well as mutations, these indications require further analysis to indicate the detection of HERG sensitive alleles.

Probes for the HERG allele can be derived from the sequence of the HERG region, its cDNA, functional equivalent sequence, or their complement. The probe may be of any suitable length spanning all or a portion of the HERG region, which allows specific hybridization to the region. If the target sequence comprises a sequence identical to that of the probe, the probe may be short, for example in the range of 8-30 base pairs, since the hybrid will be relatively stable even under stringent conditions. If some mismatch is expected with the probe, i.e., if the probe is expected to hybridize to the mutant region, a longer probe that hybridizes to the target sequence with the required specificity is used. Can be used.

Probes include isolated polynucleotides attached to a label or reporter molecule and can be used to isolate other polynucleotide sequences having sequence similarity by standard methods. Techniques for preparing and labeling probes include, for example, Sa
See mbrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by using a homologous polynucleotide. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by using the degeneracy of the genetic code. Various codon substitutions may be introduced, for example, by silent changes (thus creating various restriction sites) or may optimize expression for a particular system. Introduce a mutation,
It may modify the properties of the polypeptide, possibly altering the rate of degradation or metabolism of the polypeptide.

Probes comprising the synthetic oligonucleotides or other polynucleotides of the present invention can be derived from naturally occurring or recombinant single or double stranded polynucleotides,
Alternatively, it can be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art. A portion of the polynucleotide sequence from the HERG-encoding polynucleotide sequence, having at least about 8 nucleotides, usually at least about 15 nucleotides, and less than about 9 kb, usually less than about 1.0 kb, may be used as a probe. preferable. Therefore, this definition includes 8, 12, 15, 20, 25, 40
, 60, 80, 100, 200, 300, 400 or 500 nucleotides of a probe, or any number within these values (eg, 9, 10, 11,
Probes having nucleotides of 16, 23, 30, 38, 50, 72, 121 other nucleotides) or probes having more than 500 nucleotides. The probe can also be used to determine whether mRNA encoding HERG is present in a cell or tissue. The present invention includes all novel probes having at least 8 nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3, their complements or functionally equivalent nucleic acid sequences. The present invention does not include probes that exist in the prior art. That is, the present invention includes all probes having at least 8 nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3, but does not include probes existing in the prior art.

Similar considerations and nucleotide lengths apply to primers that can be used to amplify all or part of the HERG gene. Therefore, the definitions for primers include 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 3
A primer of 00, 400, 500 nucleotides, or any number of nucleotides within these values (eg, 9, 10, 11, 16, 23, 30, 3, 3)
8, 50, 72, 121 other nucleotides), or 50
Primers having more than zero nucleotides, or any number from 500 to 9000 nucleotides, are included. This primer can also be used to determine whether mRNA encoding HERG is present in a cell or tissue. The present invention includes all novel primers having at least 8 nucleotides from the HERG locus, their complements or functionally equivalent nucleic acid sequences for amplifying the HERG gene. The present invention does not include the primers present in the prior art. That is, the invention includes all primers having at least 8 nucleotides, but does not include primers present in the prior art.

A “protein modification or fragment” is substantially homologous to a primary structural sequence.
However, for example, in vivo or in vitro chemistry and
HERG Peptides Containing Biochemical Modifications or Incorporating Unusual Amino Acids
Provided by the present invention with respect to or a fragment thereof. Such modifications include:
For example, acetylation, carboxyl, which will be readily recognized by those skilled in the art.
, Phosphorylation, glycosylation, ubiquitination, such as labeling with radionuclides
Include various enzymatic modifications. Various methods for labeling polypeptides
Various substitutions or labels useful for various purposes are well known in the art, ³² Radioisotopes such as P, ligands that bind to labeled antiligands (eg
For example, antibodies), fluorophores, chemiluminescent agents, enzymes, and labeled ligands
Anti-ligands that can act as specific binding pair members for the same are included. Sign
Selection of the required sensitivity, ease of conjugation with primers, and stability
And the available equipment. Methods for labeling polypeptides are described in the art.
Well known in the field. See Sambrook et al., 1989 or Ausubel et al., 1992.
Hello.

In addition to substantially full-length polypeptides, the invention provides biologically active fragments of the polypeptide. Important biological activities include ligand binding, immunological and other biological activities characteristic of HERG polypeptides. Immunological activity includes both immunological function in the target immune system, as well as the shared immunological epitope to bind, acting as either a competitor or a replacement antigen for the epitope on the HERG protein. Is included. “Epitope” as used herein refers to an antigenic determinant of a polypeptide. An epitope may include three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids, and more usually, at least 8-10 such amino acids. Methods for determining the spatial conformation of such amino acids are known in the art.

For immunological purposes, tandem-repeat polypeptide segments can be used as immunogens, thereby making very antigenic proteins. Alternatively, such polypeptides will act as very efficient competitors for specific binding. The generation of antibodies specific for a HERG polypeptide or a fragment thereof is described below. The present invention also provides fusion polypeptides comprising HERG polypeptides and fragments. A homologous polypeptide may be a fusion between two or more HERG polypeptide sequences or between a HERG and a related protein sequence. Similarly, heterologous fusions may be constructed which will exhibit a combination of properties or activities of the inducible protein. For example, ligand-binding or other domains may "swap" between different new fusion polypeptides or fragments. Such homologous or heterologous fusion polypeptides can, for example, exhibit altered strength or specificity binding. Fusion partners include immunoglobulin, bacterial β-galactosidase, trpE
, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See Godowski et al., 1988. Fusion proteins can typically be made by any of the recombinant nucleic acid methods described below, or can be chemically synthesized. Techniques for synthesizing polypeptides are described, for example, in Merrifield, 1963.

“Protein purification” refers to various methods for isolating HERG polypeptide from other biological materials, such as from cells transformed with a recombinant nucleic acid encoding HERG, and is understood in the art. well known. For example, such polypeptides can be purified by immunoaffinity chromatography using, for example, the antibodies provided by the invention. Various methods of protein purification are well known in the art and include those described in Deutscher, 1990 and Scopes, 1982. The terms “isolated,” “substantially pure,” and “substantially homogeneous” are used interchangeably to describe a protein or polypeptide that has been separated from its associated components in its natural state. Used for Monomeric
A protein is substantially pure if at least about 60-75% of the sample displays a single polypeptide sequence. A substantially pure protein is typically
About 60-90% W / W protein sample, more usually more than about 95%,
And preferably more than about 99% pure. Protein purity or homogeneity can be indicated by a number of methods well known in the art, such as the visualization of a single polypeptide band during gel staining following polyacrylamide gel electrophoresis of a protein sample. . For certain purposes, higher resolution can be obtained by using HPLC or other methods well known in the art used for purification.

A HERG protein is substantially free of naturally related components when it is separated from its natural contaminants with its natural contaminants. Thus, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cells from which it naturally originates will be substantially free of its naturally related components. Proteins can be rendered substantially free of naturally related components by isolation using protein purification techniques well known in the art. A polypeptide that is isolated and produced as an expression product of a manipulated gene sequence, even when expressed in a homologous cell type, is an "isolated polypeptide" as used herein. Molecules produced synthetically or expressed by heterologous cells are naturally isolated molecules.

A “recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has been produced by the artificial combination of two other discrete segments of a sequence. This artificial combination
Frequently, either by chemical synthesis or by artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques. Such is typically done to replace codons with degenerate codons encoding the same or conservative amino acids, while typically introducing or removing sequence recognition sites. Alternatively, it is done to join together nucleic acid segments of a desired function to produce a desired combination of functions.

“Regulatory sequence” usually refers to a sequence within 100 bp of the coding sequence of a locus, but it may be further away from the coding region, including (transcription of genes and messenger RNAs). Including translation, splicing, stability, etc.)
Affects gene expression. "Substantial homology or similarity" When aligned optimally (with appropriate nucleotide insertions or deletions) with other nucleic acids (or their complements), at least about 60% of the nucleotide bases, usually At least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 9%
A nucleic acid or fragment thereof is "substantially homologous" to another when there is nucleotide sequence identity between 5-98% of the nucleotide bases (""
Or substantially similar "). To determine the homology between two different nucleic acids, the BLASTN program"
The% homology is determined using "BLAST 2 sequences."
Available for public use on the Internet from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/gorf/bl2.html)
Altschul et al., 1997). The parameters to use are the default parameters in parentheses, which combination gives the highest calculated% homology:

Program-blastn matrix-0 BLOSUM62 Rewards for matching-0 or 1 (1) Penalty for mismatch -0, -1, -2 or -3 (-2) Open gap penalty-0, 1, 2, 3, 4 or 5 (5) Extension gap penalty -0 or 1 (1) Gap x drop-off -0 or 50 (50) Forecast -10

Along with a variety of other results, the program shows% identity across the entire strand or pair of nucleic acid regions. The program shows the alignment and identity of the two strands to compare as part of the result. If the strands are of the same length, identity will be calculated across the full length of the nucleic acid. If the strands are of unequal length, the shorter nucleic acid length should be used. If the nucleic acids traverse portions of their sequence in exactly the same way, but traverse the rest of their sequences differently, the blastn program "BLAST2 Sequences" will show identity only over similar portions and these portions will be reported separately. Is done. For purposes of determining homology herein,% homology refers to the shorter of the two sequences being compared.
If any one region is represented by a different alignment with different% identity,
The alignment that gives the highest homology should be used. Averaging, SEQ ID NO:
5 and 6 as in this example.

[0088]

Embedded image

The program “BLAST 2 Sequences” shows different alignments of these two nucleic acids depending on the parameters selected. For example, four sets of parameters were selected to compare SEQ ID NOs: 5 and 6 (gap x dropoff was 50 for all cases) and the results are shown in Table 1.

[0090]

[Table 1]

It should be noted that any of the set of parameters selected as shown in Table 1 is not necessarily the best set of parameters for comparing these sequences. The percent homology is calculated by multiplying the fraction of bases of the shorter strand in the region by the percent identity for that region for each region showing identity, and adding them all together. For example, using the first set of parameters shown in Table 1, SEQ ID NO: 5 is the shorter sequence (63 bases), indicating a region of 2 identity. The first one has 92% identity to SEQ ID NO: 6.
5 bases 4-29 (26 bases), the second comprising 10 to SEQ ID NO: 6
Includes bases 39-59 (21 bases) of SEQ ID NO: 5 with 0% identity. Base 1
-3, 30-38 and 60-63 (16 bases) are not shown to have any identity to SEQ ID NO: 6. The% homology is: (26/63) (92) + (2
Calculated as 1/63) (100) + (16/63) (0) = 71.3% homology. Table 1 lists the percent homology calculated using each of the four sets of parameters shown. Several other parameter combinations are possible, but they are not listed for brevity. It is shown that the parameters of each set have different calculated% homology. Only the results that give the highest% homology should be used,
Based on the parameters of the set, SEQ ID NOs: 5 and 6 will have 87.1% homology. Again, using other parameters, SEQ ID NOs: 5 and 6
May show higher homology, but for the sake of brevity, not all possible results are shown.

Alternatively, if under selective hybridization conditions the nucleic acid or fragment thereof will hybridize to another nucleic acid (or its complement), a substantial change in the strand or its complement will occur. There is homology or (similarity). Hybridization selectivity exists when hybridization occurs that is substantially more selective than the overall lack of specificity. Typically,
At least about 55% over a stretch of at least about 14 nucleotides,
Preferably hybridization will occur if there is preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% homology. See Kanehisa, 1984. The length of the homology comparison can span a longer stretch, as described, and in certain embodiments, is at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically It will often span a stretch of at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

[0093] Nucleic acid hybridization, as readily understood by those skilled in the art, is based on salt concentration, temperature, as well as base composition, complementary strand length, and the number of nucleotide base mismatches between hybridized nucleic acids. Or conditions such as organic solvents. Stringent temperature conditions generally include temperatures above 30 ° C,
Typically temperatures above 37 ° C, and preferably above 45 ° C, will be included. Stringent salt concentrations will usually be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM.
However, the combination of parameters is much more important than the measurement of any single parameter. Stringency conditions will depend on the length of the nucleic acid and the base composition of the nucleic acid, and may be determined by techniques well known in the art. We
See tmur and Davidson, 1968.

[0094] Probe sequences can also specifically hybridize to duplex DNA under certain conditions to form triplexes or other higher dimensional DNA complexes. The preparation of such probes and suitable hybridization conditions are well known in the art.

The terms “substantially homologous” or “substantially identical,” when referring to a polypeptide, refer to the polypeptide or protein of interest as a whole or at least about a naturally occurring protein or portion thereof. 30% identical, usually at least about 70% identical, more usually at least about 80% identical, preferably at least about 9%
0% identical, and more preferably at least about 95% identical. Homology for polypeptides is typically measured using sequence analysis software. For example, Genetics Computer Group, University of Wisconsin Biote
Sequen at chnology Center, 910 University Avenue, Madson, Wisconsin 53705
See ce Analysis Software Package. Protein analysis software matches similar sequences using measures of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include the following groups of substitutions:
Glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

“Substantially similar function” refers to the function of a modified nucleic acid or protein with reference to a wild-type HERG nucleic acid or a wild-type HERG polypeptide. The modified polypeptide will be substantially homologous to the wild-type HERG polypeptide and will have substantially the same function. Modified polypeptides can have an altered amino acid sequence and / or can include modified amino acids. In addition to similarity in function, the modified polypeptides may have other useful properties, such as a longer half-life. The similarity of function (activity) of the modified polypeptide can be substantially the same as that of the wild-type HERG polypeptide. Alternatively, the similarity of function (activity) of the modified polypeptide may be higher than that of the wild-type HERG polypeptide. Modified polypeptides are synthesized using conventional techniques, or are encoded by modified nucleic acids and produced using conventional techniques. Modified nucleic acids are prepared by conventional techniques. Nucleic acids having functions substantially similar to wild-type HERG gene function produce the modified proteins described above.

A polypeptide “fragment”, “portion” or “segment” is at least about 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids And most preferably a stretch of amino acid residues of at least about 20 to 30 or more contiguous amino acids. The polypeptides of the present invention, when soluble, can be used on solid supports such as nitrocellulose, nylon, column packing (eg, Sepharose beads), magnetic beads, glass wool, plastics, metals, polymers. It can be coupled to a gel, cell or other substrate. Such supports may take the form of, for example, beads, wells, dipsticks or membranes.

“Target region” refers to a region of a nucleic acid to be amplified and / or detected. The term "target sequence" refers to a sequence with which a probe or primer will form a stable hybrid under the conditions of interest. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunology. For example, Maniatis et al., 1982;
See Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991. A general discussion of techniques and materials for human gene mapping, including mapping of human chromosome 1, can be found, for example, in White and Lalouel, 19
88 are provided.

Preparation of Recombinant or Chemically Synthetic Nucleic Acids; Vectors, Transformations, Preparation of Host Cells Large quantities of the polynucleotides of the invention may be produced by replication in a suitable host cell. The natural or synthetic polynucleotide fragment encoding the fragment of interest is introduced into a recombinant polynucleotide construct, usually a DNA construct, which can be introduced into prokaryotic or eukaryotic cells and replicated therein. Normal,
The polynucleotide construct will be suitable for replication in a unicellular host such as a yeast or bacterium, but will be introduced (with or without integration into the genome) into a cultured mammal or plant or other eukaryotic cell line. May also be intended. Purification of nucleic acids produced by the methods of the invention is described, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may be prepared by the phosphoramidite method described by Beaucage and Caruthers, 1981 or by Matteucci and Caruthers, 1981.
Can be produced by a chemical synthesis by the triester method according to the above, and can be performed by a commercially available automated oligonucleotide synthesizer. Double stranded fragments are chemically synthesized by either synthesizing the complementary strand and annealing the strands together under appropriate conditions, or adding the complementary strand using a suitable primer sequence and DNA polymerase. It can be obtained from single-stranded products.

A polynucleotide construct prepared for introduction into a prokaryotic or eukaryotic host contains the intended polynucleotide fragment, which encodes the polypeptide of interest, and is preferably operable on a segment which encodes the polypeptide. And may include a replication system recognized by the host that will also include transcriptional and translational initiation regulatory sequences linked to. Expression vectors include, for example, an origin of replication or an autonomously replicating sequence (A
RS) and necessary processing information such as expression control sequences, promoters, enhancers, and ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilization sequences.
Such vectors are well known in the art and are described, for example, in Sambrook et al., 19
89 or Ausubel et al., 1992, by standard recombinant techniques.

[0102] Appropriate vectors and other necessary vector sequences are selected to function in the host and, where appropriate, may include those naturally associated with the HERG gene. Examples of operable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or
Ausubel et al., 1992; see also Metzger et al., 1988. Many useful vectors are known in the art and include Stratagene, New England
It can be obtained from vendors such as Biolabs and Promega Biotech. trp,
Promoters such as lac and phage promoters, tRNA promoters and glycolytic enzyme promoters can be used in prokaryotic hosts. Useful yeast promoters include promoter regions for other glycolytic enzymes such as metallothionein, 3-phosphoglycerate kinase or enolase or glyceraldehyde 3-phosphate dehydrogenase, enzymes that contribute to maltose and galactose utilization, and the like. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Suitable non-natural mammalian promoters include the early and late promoters from SV40 (Fiers et al., 197).
8) or promoters from Moloney leukemia virus, mouse tumor virus, chicken sarcoma virus, adenovirus II, bovine papilloma virus or polyoma may be included. The insect promoter may be from a baculovirus. In addition, the construct can bind to an amplifiable gene (eg, DHFR) so that multiple copies of the gene can be generated. For suitable enhancers and other expression control sequences, see Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press.
, Cold Spring Harbor, New York (1983). See also, for example, U.S. Patent Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.

While such expression vectors can replicate autonomously, they can also replicate by inserting into the genome of a host cell, by methods well known in the art. Expression and cloning vectors are likely to include a selectable marker, a gene encoding a protein necessary for the survival or growth of the host cell transformed with the vector. The presence of this gene ensures that only host cells expressing the insert will grow. Typical selection genes are: a) conferring resistance to antibiotics or other toxic substances such as ampicillin, neomachine, methotrexate, etc., b) supplementing auxotrophic deficiencies, or c) essential nutrients not available from complex media Supply (
For example, Bacilli encodes D-alanine racemase) and a protein. Selection of the appropriate selectable marker will depend on the host cell, and appropriate markers for different hosts are well-known in the art.

The vector containing the nucleic acid of interest is transcribed in vitro and the resulting RNA is transformed into host cells by well known techniques, such as injection (see Kubo et al., 1988). Or electroporation; calcium chloride, rubidium chloride, calcium phosphate, DEAE-
Depends on the type of cell host, including transfection with dextran or other substances; microprojectile bombardment; lipofection; infection (if the vector is an infectious agent such as the retroviral genome); and other methods. And can be introduced directly into host cells by methods well known in the art. See, generally, Sambrook et al., 1989 and Ausubel et al., 1992. Introduction of a polynucleotide into a host cell by any method known in the art, including, inter alia, those described above, is referred to herein as "transformation." Cells into which the nucleic acid has been introduced,
It is meant to include the progeny of such cells.

[0105] Large quantities of the nucleic acids and polynucleotides of the invention can be prepared by expressing the HERG nucleic acid or a portion thereof in a compatible prokaryotic or eukaryotic host cell in a vector or other expression carrier. Bacills
Other prokaryotes such as P. us subtilis or Pseudomonas can be used, but the most commonly used prokaryotic host is Escherichia coli (Es
cherichia. coli) strain.

Mammalian or other eukaryotic host cells, such as yeast, mold, plant, insect or amphibian or bird species, may also be useful for producing the proteins of the invention.
Propagation of mammalian cells in culture is well known per se. Jakoby and Pa
See Stan, ed., 1979. It will be appreciated by those skilled in the art that other cell lines may be suitable, for example, to provide higher expressivity, desirable glycosylation patterns or other characteristics, but examples of commonly used mammalian cell lines are: VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK and COS cell lines. An example of a commonly used insect cell line is SF9.

[0107] Clones are selected by using a marker that depends on the mode of the vector construct. The markers can be on the same or different DNA molecules, preferably on the same DNA molecule. In prokaryotic hosts, transformants may be selected for example by resistance to ampicillin, tetracycline or other antibiotics. The production of certain products based on temperature sensitivity can also serve as a suitable marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only in producing the nucleic acids and polypeptides of the present invention, but also in, for example, studying the characteristics of HERG polypeptides. . Probes and primers based on the HERG gene sequences disclosed herein are used to identify homologous HERG gene sequences and proteins of other species. These gene sequences and proteins are used in the diagnostic / prognostic, therapeutic and drug screening methods described herein for the species from which they were isolated.

Methods of Use: Drug Screening The present invention is particularly useful for screening compounds by using HERG polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The HERG polypeptide or fragment thereof used in such a test can be either free in solution, fixed to a solid support, or carried to the cell surface. One method of drug screening utilizes a eukaryotic or prokaryotic host cell stably transformed with a recombinant polynucleotide expressing the polypeptide or fragment, preferably in a competitive binding assay. Such cells, in variable or fixed form, can be used for standard binding assays. For example, the formation of a complex between a HERG polypeptide or fragment and an agent to be tested can be measured, or the formation of a complex between a HERG polypeptide or fragment and a known ligand can be measured. Can determine the degree of disturbance.

Thus, the present invention provides for contacting such an agent with a HERG polypeptide or fragment thereof, and by contacting the agent with a HERG polypeptide or fragment by methods well known in the art. A method of screening for an agent comprising assaying for the presence of a complex or (ii) for the presence of a complex between a HERG polypeptide or fragment and a ligand is provided.
In such competitive binding assays, the HERG polypeptide or fragment is typically labeled. Free HERG polypeptide or fragment is separated from that present in the protein: protein complex, and the amount of free (ie, uncomplexed) label is determined by the amount of binding of the agent to be tested to HERG or HER, respectively.
G: A measure of its interference with ligand binding. Also, the amount of bound HERG, rather than free, can be measured. It is also possible to label the ligand, rather than HERG, and determine the amount of ligand bound to HERG in the presence or absence of the drug to be tested.

Another technique for drug screening provides high-throughput screening for compounds with suitable binding affinity for HERG polypeptides,
en (International Publication WO84 / 03564). Briefly, a number of different small peptide test compounds are synthesized on a solid substrate, such as a plastic pin or other surface. The peptide test compound is reacted with the HERG polypeptide and washed. The bound HERG polypeptide is then detected by methods well known in the art.

[0112] Purified HERG can be coated directly onto a plate for use in the drug screening techniques described above. However, the antibody can be captured using a non-neutralizing antibody against the polypeptide and HERG polypeptide immobilized on a solid phase. The present invention also contemplates the use of competitive drug screening assays in which a neutralizing antibody capable of specifically binding to a HERG polypeptide competes with a test compound that binds to a HERG polypeptide or fragment thereof. In this way,
Antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants of a HERG polypeptide. The screening method is not limited to assays using HERG alone, but is also applicable to study HERG-protein complexes. The effect of the drug on the activity of this complex is analyzed.

In accordance with these methods, the following assays are examples of assays that can be used to screen drug candidates. The mutant HERG (by itself or as part of a fusion protein) is mixed with the wild-type protein to which wild-type HERG binds (by itself or as part of the fusion protein). This mixing is performed both in the presence of the drug and in the absence of the drug, and the amount of binding between the mutant HERG and the wild-type protein is measured. If the amount of binding is greater in the presence of the drug than in the absence of the drug, the drug is HE
Drug candidate for treating LQT resulting from mutations in RG.

The wild-type HERG (as itself or as part of a fusion protein) is mixed with the wild-type protein to which wild-type HERG binds (either by itself or as part of the fusion protein). This mixing is performed both in the presence of the drug and in the absence of the drug, and the amount of binding between the wild-type HERG and the wild-type protein is measured. If the amount of binding is greater in the presence of the drug than in the absence of the drug, the drug will bind to HERG
Drug candidate for treating LQT resulting from mutations in it.

A mutein as a wild-type protein that binds to HERG (itself or as part of a fusion protein) is mixed with wild-type HERG (itself or as part of a fusion protein). This mixing is performed both in the presence of the drug and in the absence of the drug, and the amount of binding between the mutant protein and wild-type HERG is measured. If the amount of binding is greater in the presence of the drug than in the absence of the drug, the drug is a drug candidate for treating LQT resulting from a mutation in the gene encoding the protein. .

The polypeptides of the present invention may also be used for screening compounds developed as a result of combinatorial library technology. Combinatorial library technology offers an efficient way to test a potentially vast number of different substances for their ability to modulate the activity of a polypeptide. Such libraries and their uses are known in the art. The use of a peptide library is preferred.
See, for example, WO 97/02048. Briefly, methods for screening for substances that modulate the activity of a polypeptide include contacting one or more test substances with the polypeptide in a suitable reaction medium, measuring the activity of the treated polypeptide, It may include comparing the activity to the activity of the polypeptide in a comparable reaction medium that has not been treated with the test substance or substances. Differences in activity between the treated and untreated polypeptides are indicative of a modulating effect of the relevant test substance or substances. Before and during screening for modulation of activity, test substances may be used, for example, in a yeast two-hybrid system (eg, Bartle et al., 1993; Fields and
Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995) may be screened for the ability to interact with a polypeptide. This system can be used as a rough screen before testing substances for the actual ability to modulate the activity of the polypeptide. Alternatively, the screen could be used to screen test substances for binding to a HERG-specific binding partner, such as myosin, actin or dystrophin, or to find a mimic of a HERG polypeptide.

After identifying a substance that modulates or affects polypeptide activity, the substance can be further examined. Further, it may be manufactured and / or used in a preparation, ie, a product or formulation, or a composition, such as a medicament, pharmaceutical composition or medicament. These can be administered to individuals.

Thus, the present invention is directed not only to substances identified using nucleic acid molecules as modulators of polypeptide activity, but also to pharmaceutical compositions, medicaments comprising such substances, consistent with those disclosed herein. A method comprising administering such a composition comprising a drug or other composition, such a substance, treating such a composition, eg, LQT (
Including administering prophylactic treatment) to a patient, eg, LQT
And the use of such substances in the manufacture of compositions to be administered, and the mixing of such substances with pharmaceutically acceptable excipients, vehicles or carriers, and optionally other ingredients, to treat The present invention covers various embodiments including a method for producing the composition.

[0119] Substances identified as modulators of polypeptide function can be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical applications. Thus, mimics or mimetics of the substance (especially in the case of peptides) can be designed for pharmaceutical use. The design of mimetics for known pharmaceutically active compounds is a known approach to the development of drugs based on "lead" compounds. This may be the case if the active compound is difficult or expensive to synthesize, or if it is not suitable for a particular mode of administration (for example, a pure peptide may be rapidly degraded by proteases in the gastrointestinal tract). This is desirable because it is an active agent that is often not suitable for oral compositions). Mimic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for target properties.

There are several steps commonly taken to design a mimetic from a compound having a given target property. First, determine the specific portions of the compound that are critical and / or important in determining the target properties. In the case of peptides, this can be done by altering the amino acid residues in the peptide entirely, eg, by sequentially substituting each residue. An alanine scan of the peptide is commonly used to clarify such a peptide motif. These moieties or residues that make up the active region of the compound are referred to as their “pharma cocoa”
Also known as

Once a pharma cocoa has been found, its physical properties, such as stereochemistry, connectivity, size and / or using data from a range from the source, eg, spectroscopic techniques, x-ray diffraction data and NMR. Model the structure according to the charge. Computer analysis, similar mappings (modeling the charge and / or volume of pharmaccocoa rather than bonds between atoms) and other techniques can be used in this modeling step. In a variation on this approach, the three-dimensional structure of the ligand and its binding partner is modeled. This allows a model in which the ligand and / or binding partner changes conformation upon binding and takes this into account in mimetic design.

Next, a template molecule to which a chemical group mimicking the pharmaccocoa is to be implanted is selected.
The template molecule and its grafted chemical groups should be such that the mimetic is easy to synthesize, appears to be pharmacologically acceptable, and is not degraded in vivo, but retains the biological activity of the lead compound. , Can be routinely selected. Alternatively, where the mimetic is based on a peptide, additional stability may be achieved by cyclizing the peptide and increasing its rigidity. The mimetics or mimetics found by this approach can then be screened to understand if they have target properties or to what extent they show it. Additional optimizations or modifications may then be made to arrive at one or more final mimics for in vivo or clinical trials.

Methods of Use: Nucleic Acid Diagnostics and Diagnostic Kits To detect the presence of a HERG allele that predisposes an individual to LQT, a biological sample such as blood is prepared and analyzed for the presence or absence of a susceptibility allele of HERG. . Biological samples were prepared and analyzed for the presence or absence of the mutant allele of HERG to detect its presence as an LQT or prognostic indicator. The results and interpretation information of these tests will be returned to the healthcare provider for communication to the test individuals. Such diagnosis may be performed by a diagnostic laboratory, or, alternatively, a diagnostic kit may be manufactured and sold to a healthcare provider or an individual for self-diagnosis. First, the screening method involves amplification of the relevant HERG sequence. In another preferred embodiment of the invention, the screening method comprises a non-PCR based strategy. Such screening methods include two-step label amplification methods well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

The most familiar method used today is target amplification. Here, the target nucleic acid sequence is amplified using a polymerase. One particularly preferred method using polymerase-operated amplification is the polymerase chain reaction (PCR). The polymerase chain reaction and other polymerase-operated amplification assays can achieve over one million-fold increase in copy number through the use of polymerase-operated amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for a DNA probe. If a probe is used to detect the presence of the target sequence, the biological sample to be analyzed, such as blood or serum, can be processed and, if desired, the nucleic acid extracted. The sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence, for example, denaturation, restriction digestion, electrophoresis or dot blotting. The target region of the analyte nucleic acid must usually be at least partially single-stranded and hybridize to the target sequence of the probe. If the sequence is naturally single-stranded, no denaturation will be necessary. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be performed by various methods known in the art.

The analyte nucleic acid and the probe are incubated under conditions that promote stable hybridization of the target sequence in the probe with the putative evaluation sequence in the analyte. The region of the probe used to bind the analyte can be completely complementary to the targeted region of human chromosome 7. Therefore, high stringency conditions are desirable to prevent false positives. However, high stringency conditions are only used if the probe is complementary to a region of the chromosome that is unique to the genome. The stringency of hybridization depends on many factors during the hybridization and washing steps, including temperature, ionic strength, base composition, probe length, and formamide concentration. These factors are described, for example, in Maniatis et al.
1982 and Sambrook et al., 1989. Under certain circumstances, the formation of higher order hybrids, such as triplexes, quadraplexes, etc., may be desirable to provide a method for detecting a target sequence.

In any case, the detection of the resulting hybrid is usually carried out by using a labeled probe. Alternatively, the probe may be unlabeled, but may be detected by specifically binding directly or indirectly to the labeled ligand. Suitable labels and methods for labeling probes and ligands are known in the art and include, for example, known methods (eg, nick translation, random
Radioactive labels, biotin, fluorescent groups, chemiluminescent groups (eg, dioxetanes, especially activated dioxetanes), enzymes, antibodies, gold nanoparticles, and the like, which can be incorporated by priming or kinasing) are included. Variations of this basic scheme are known in the art and include various variations that facilitate the separation of the hybrid to be detected from those that amplify the signal from foreign materials and / or labeling groups. A number of these variants are described, for example, in Matthews and Kricka, 1988.
Landegren et al., 1988; Mifflin, 1989; U.S. Pat. No. 4,868,105; and EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated by the present invention. This approach allows nucleic acid probes (or analogs such as methyl phosphonate to replace the normal phosphodiester) to hybridize to low levels of DNA targets. The probe may have an enzyme covalently attached to the probe such that the covalent attachment does not interfere with the specificity of the hybridization. Then, this enzyme-
The probe-conjugate-target nucleic acid complex is isolated from the probe-free enzyme conjugate and the substrate is added for enzyme detection. Enzyme activity is 1 with sensitivity
Observed as a change in color or luminescent output resulting in an increase of 0 ³ -10 ⁶ . See Jablonski et al., 1986 for examples on the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

[0128] Two-step label amplification methods are known in the art. These assays work on the principle that small ligands (such as digoxigenin, biotin, etc.) bind to nucleic acid probes that can specifically bind HERG. Allele-specific probes are also contemplated within the scope of this example, and exemplary allele-specific probes include probes that include a predisposition mutation of this disclosure. In one example, the small ligand bound to the nucleic acid probe is specifically recognized by the antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to a nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate that metabolizes the chemiluminescent substrate. For the method of labeling a nucleic acid probe according to this embodiment, for example, Ma
See rtin et al., 1990. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that can specifically complex with the first ligand. A well-known embodiment of this example is the biotin-avidin type interaction. For methods for labeling nucleic acid probes and biotin-avidin based assays see Rigby et al., 1977 and Nguyen et al., 1992.

It is also contemplated within the spirit of the present invention that the nucleic acid probe assays of the invention will use a cocktail of nucleic acid probes capable of detecting HERG. Thus, in one example for detecting the presence of HERG in a cell sample, more than one probe complementary to the gene is used, in particular, the number of different probes is alternatively 2
3 or 5 different nucleic acid probe sequences. In another example, more than one probe complementary to these genes is used to detect the presence of a mutation in the patient's HERG gene sequence, wherein the cocktail is a population of patients having a change in HERG Probes that can bind to the allele-specific mutation identified therein. In this embodiment, any number of probes may be used, and will preferably include probes corresponding to oncogene mutations identified as contributing to an individual's predisposition to LQT.

Methods of Use: Peptide Diagnosis and Diagnostic Kits The presence of LQT can also be detected based on changes in wild-type HERG polypeptide. Such changes may be determined by sequence analysis by conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in HERG peptides or the absence of HERG peptides. Techniques for raising and purifying antibodies are well known in the art, and any such technique may be selected to achieve the preparations claimed in the present invention. In a preferred embodiment of the invention, the antibodies will immunoprecipitate HERG proteins from solution and will react with these proteins on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, using immunohistochemical techniques, the antibodies will detect HERG protein in paraffin or frozen tissue sections. Preferred embodiments relating to the method of detecting HERG or mutations thereof include sandwich assays using monoclonal and / or polyclonal antibodies, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIAs), immunoradiometric assays ( IRMA) and immunoenzymatic assay (IEMA). Exemplary sandwich assays are described in U.S. Patent Nos. 4,376,110 and 4,486,530, which are hereby incorporated by reference.
Described by David et al.

Methods of Use: Rational Drug Design The goal of rational drug design is, for example, a more active or stable form of the polypeptide, or for example, to enhance the function of the polypeptide in vivo. The purpose is to create a structural analog of the biologically active polypeptide of interest or a small molecule (eg, agonist, antagonist, inhibitor) that interacts with it for the purpose of forming an interfering drug. For example, Hodg
See son et al., 1991. In one approach, first the three-dimensional structure of a protein of interest (eg, a HERG polypeptide) is determined by x-ray crystallography, by computer modeling, or most typically by a combinatorial approach. Less often, useful information about the structure of a polypeptide can be obtained by modeling based on the structure of homologous proteins. An example of a rational drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides (eg, HER
G polypeptide) is analyzed by alanine scan (Wells, 1991). In this technique, an amino acid residue is replaced with Ala and its effect on the activity of the peptide is determined. Each amino acid residue of the peptide is analyzed in this way to determine important regions of the peptide.

It is also possible to isolate a target-specific antibody selected by a functional assay and then resolve its crystal structure. In principle, this approach provides a pharma cocoa on which secondary drug design can be based. By generating anti-idio antibodies (anti-ids) to functional, pharmacologically active antibodies,
It is possible to bypass protein crystallography altogether. Anti-id as mirror image of mirror image
The binding site for s would be expected to be an analog of the original receptor. Then anti-ids
Can be used to identify and isolate peptides from chemically or biologically generated banks of peptides. The selected peptide will then act as a pharmacore.

Thus, for example, one can design drugs that have improved HERG polypeptide activity or stability, or that act as inhibitors, agonists, antagonists, and the like of HERG polypeptide activity. Due to the availability of cloned HERG sequences, a sufficient amount of HER useful for conducting analytical studies such as x-ray crystallography
G polypeptides can be made. In addition, the knowledge of the HERG protein sequence described herein will guide the use of computer modeling techniques instead of or in addition to x-ray crystallography.

Method of Use: Gene Therapy In accordance with the present invention, wild-type HERG is added to cells carrying a mutant HERG allele.
A method for providing a function is also provided. Providing such a function would allow normalization of the recipient cells. A wild-type gene or a portion of a gene can be introduced into cells with a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from an extrachromosomal location. More preferably, the situation is such that the wild-type gene or a part thereof is introduced into the mutant cell such that the endogenous mutant gene present in the cell and the wild-type gene or a part thereof are recombined. Such recombination requires a double recombination event resulting in correction of the gene mutation. Vectors for introducing both recombinant and extrachromosomal genes are known in the art, and any suitable vector may be used. Methods for introducing DNA into cells, such as electroporation, calcium phosphate co-precipitation and virus introduction, are known in the art, and the choice of method is within the capabilities of the practitioner.

As discussed generally above, the HERG gene or fragment, when applicable, may be used to increase the amount of the expression product of such a gene in a cell,
Can be used for gene therapy. Also, it can be useful to increase the level of expression of a given LQT gene, even in heart cells where the mutant gene is expressed at "normal" levels but the gene product is not fully functional. Gene therapy can be performed according to generally accepted methods, for example, Friedman (19)
91) or as described by Culver (1996).
Cells from the patient are first analyzed by the diagnostic method described above to determine the HER in the cells.
It will confirm the production of the G polypeptide. HE linked to expression control elements
A virus or plasmid vector containing a copy of the RG gene and capable of replicating inside the cell (see further details below) is prepared. Vectors can replicate inside cells. Alternatively, the vector can be replication deficient, which is inherited and replicates in helper cells for use in therapy. U.S. Pat. No. 5,252,479 and WO 98/07282 and U.S. Pat.
Suitable vectors are known, such as disclosed in 5,691,198; 5,747,469; 5,436,146 and 5,753,500. The vector is then injected into the patient. If the transfected gene is not permanently integrated into the genome of each target cell, the treatment must be repeated periodically.

[0136] Gene transfer systems known in the art can be useful for performing the gene therapy of the present invention. These include viral and non-viral transfer methods. A variety of viruses have been used as a transfer vector or as a basis for preparing a transfer vector, including papovaviruses (eg, SV40, Ma
dzak et al., 1992), adenovirus (Berker, 1992; Berkner et al., 1988; Gorziglia).
And Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson and Akrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al., 1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus ( Muzyczka
Ohi et al., 1990; Russell and Hirata, 1998), herpes viruses including HSV and EBV (Mardolskee, 1992; Johnson et al., 1992; Fink et al., 1992; B).
reakefield and Geller, 1987; Freese et al., 1990; Fink et al., 1996), lentivirus (Naldini et al., 1996), Sindbis and Semliki forest virus (Berglund).
Et al., 1993), and birds (Bandyopadhyay and Temin, 1984; Petropoulos).
Et al., 1992); rodents (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Man).
n and Baltimore, 1985; Miller et al., 1988) and of human origin (Shimada et al., 199).
1; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992) retroviruses. Most human gene therapy protocols are based on neutralized rodent retroviruses, although adenoviruses and adeno-associated viruses have also been used.

Non-viral gene transfer methods known in the art include calcium phosphate co-precipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques such as, for example, μ injection ( Anderson et al., 1980; Gordon et al., 1980; Brin
ster et al., 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991). ); And direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989).
Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel;
Curiel et al., 1991). Virus-mediated gene transfer, in combination with direct in vivo gene transfer using liposome delivery, allows the viral vector to be directed to tumor cells but not to surrounding non-dividing cells. Alternatively, a retroviral vector producer cell line may be injected into the tumor (Culver et al., 1992). In that case, injection of the producer cells will provide a continuous source of vector particles. This technique has been approved for use in patients with inoperable brain tumors.

In an approach that combines biological and physical gene transfer, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific for adenovirus hexon protein. The resulting complex is ligated to an adenovirus vector. In this case, the cells are infected using the trimolecular complex. Adenovirus vectors allow efficient binding, internalization, and degradation of endosomes before the coupled DNA is damaged.
For other techniques for delivering adenovirus-based vectors, see Schnei.
See der et al. (1998) and U.S. Patent Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.

It has been shown that liposome / DNA complexes can mediate in vivo gene transfer directly. Inherited transfer processes are non-specific in standard liposome preparations, but localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992)
. An expression vector in the context of gene therapy is meant to include a construct containing sequences that sufficiently express the cloned polynucleotide therein. In a viral expression vector, the construct contains sufficient viral sequences to support packaging of the construct. If the polynucleotide encodes HERG, expression will produce HERG. If the polynucleotide encodes an antisense polynucleotide or ribozyme, expression will produce an antisense polynucleotide or ribozyme.
Thus, in this context, expression does not require that the protein be synthesized. In addition to the polynucleotide cloned into the expression vector, the vector also includes a promoter function in eukaryotic cells. The cloned polynucleotide sequence is under the control of this promoter. Suitable eukaryotic promoters include those described above. Expression vectors can also include sequences such as the selectable markers and other sequences described herein.

[0140] Gene transfer techniques that target DNA directly to heart tissue are preferred. Receptor-mediated gene transfer is performed, for example, by conjugating DNA (usually in the form of a covalently supercoiled plasmid) to the protein ligand via polylysine. Ligands are selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell / tissue type. These ligand-DNA conjugates can be injected directly into the blood, if desired, and directed to the target tissue where receptor binding and internalization of the DNA-protein conjugate occurs. To overcome the problem of intracellular destruction of DNA, endosomal function can be disrupted, including co-infection with adenovirus. The treatment is as follows: patients carrying the HERG sensitive allele are treated with gene delivery such that some or all of their cardiac precursor cells receive at least one additional copy of a functional normal HERG allele. I do. In this step, the treated individual has a reduced risk of LQT to the extent that the presence of the normal allele suppresses the effects of the susceptible allele.

Methods of Use: Peptide Therapy A peptide having HERG activity can be supplied to cells that carry a mutant or deleted HERG allele. The protein can be produced, for example, by expressing the cDNA sequence in bacteria using a known expression vector. Alternatively, the HERG polypeptide may be extracted from a HERG-producing mammalian cell.
In addition, HERG proteins can be synthesized using synthetic chemistry techniques. Any of such techniques can provide a preparation of the invention comprising a HERG protein.
The preparation is substantially free of other human proteins. This is easily achieved by synthesis in microorganisms or in vitro. Active HERG molecules can be introduced into cells by microinjection or by using liposomes. Alternatively, certain active molecules are
It can be taken up by cells by active or diffusion. The supply of molecules with HERG activity must lead to a partial inversion of LQT. Other molecules with HERG activity (eg, peptides, drugs or organic compounds) can also effect such reversal. Modified polypeptides having substantially similar functions are also used for peptide therapy.

Methods of Use: Transformed Hosts Animals to test the therapeutic agent can be selected after mutation of the whole animal or after treatment of germ cells or zygotes. Such treatment usually involves the insertion of mutant HERG alleles from another animal species, as well as the insertion of split homologous genes. Alternatively, the endogenous HERG gene of the animal can be obtained using the conventional techniques (Capecchi, 198).
9; Valancius and Smithies et al., 1991; Hasty et al., 1991; Shinkai et al.
1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992.
92; Donehower et al., 1992), which can be disrupted by insertion or deletion mutations or other genetic alterations. After the test substance has been administered to the animal, the presence of LQT must be assessed. If the test substance prevents or suppresses the appearance of LQT, the test substance is a candidate therapeutic for the treatment of LQT. These animal models provide a very important test vehicle for potential therapeutic products.

The identification of the association between a HERG gene mutation and LQT allows an individual's early presymptomatic screening to identify the risk for developing LQT.
To identify such individuals, HERG alleles are screened for mutation either directly or after cloning of the allele. The allele is tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, but not limited to, one of the following methods: fluorescent in situ Hybridization (FISH), direct DNA sequencing, P
FGE analysis, Southern blot analysis, single-stranded conformation analysis (SSCP
), Linkage analysis, RNase protection analysis, allele-specific oligonucleotide (A
SO), dot plot analysis and PCR-SSCP analysis. Also, a recently developed DNA microchip technology is useful. For example, (1) determining the nucleotide sequence or the appropriate fragment (coding or genomic sequence) of both the cloned allele and the normal HERG gene and then comparing, or
(2) the RNA transcript of the HERG gene or gene fragment is hybridized with single-stranded total genomic DNA from the individual to be tested, and the resulting heteroduplex is treated with ribonuclease A (RNase A) And run on a denaturing gel to detect any mismatches. Two of these methods can be performed according to the following procedure.

Alleles of the HERG gene in the individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from an individual. Genomic DNA isolated from cells in this sample is partially digested to an average fragment size of about 20 kb. Fragments ranging from 18-21 kb are isolated. The obtained fragment is
It is ligated to an appropriate vector. The clones were then sequenced and normal HE
Compared to the RG gene.

[0145] Alternatively, the polymerase chain reaction (PCR) is performed using primer pairs for the 5 'region or exons of the HERG gene. In addition, PCR may be performed using primers based on any sequence of a normal HERG gene.
For example, a primer pair of one of the introns can be prepared and utilized. Finally, RT-PCR may also be performed on the mRNA. Amplification products were analyzed by single-stranded conformational analysis (SSC) using conventional techniques to distinguish any differences.
P) to identify any differences, which are then sequenced and compared to the normal gene sequence.

Amplification of individual DNA using the use of appropriate primer pairs and analysis of amplification products, for example, by common HERG gene mutations by dot blots using oligonucleotide probes specific for the allele The body can be screened quickly.

The second method uses RNase A to help detect differences between the normal HERG gene and the defective gene. The comparison is made in a step using a small (about 500 bp) restriction fragment of the HERG gene as a probe. First, the HERG gene is digested with restriction enzymes, cutting the gene sequence into fragments of about 500 bp. These fragments are individually separated on an electrophoresis gel, purified from the gel, and cloned in both orientations into an SP6 vector (eg, pSP64 or pSP65). The SP6-based plasmid containing the insertion of the HERG gene fragment was [α- ³² P
] Transcribed in vitro using the SP6 transcription system well known in the art in the presence of GTP to obtain radiolabeled RNA transcripts of both strands of the gene.

Individually, using these RNA transcripts, allelic DNA was synthesized using conventional techniques.
To form a heteroduplex. The mismatch caused in the RNA: DNA heteroduplex due to sequence differences between the HERG fragment from the individual and the HERG allele subclone results in cleavage of the RNA strand when treated with RNaseA. Such mismatches may be the result of a point mutation or small deletion in an individual's allele. Cleavage of the RNA strand results in two or more small RNA fragments,
Runs faster on denaturing gels than the A probe itself.

[0149] Any differences found will identify molecular variants of the HERG gene, and individuals who have an inevitable presence of LQT. These variants can take many forms. Most severe forms are frameshift mutations or large deletions that cause the gene to code for an abnormal protein, or that significantly alter protein expression. Less severe destructive mutations include small in-frame deletions and non-conservative base pair substitutions, including changes to or from cysteine residues, changes from basic to acidic amino acids, and vice versa. Such changes from hydrophobic to hydrophilic amino acids or vice versa, or other mutations that affect secondary or tertiary protein structure, will have a significant effect on the protein produced. Silent mutations or those that produce conservative amino acid substitutions were generally not expected to disrupt protein function.

Pharmaceutical Compositions and Routes of Administration The HERG polypeptides, antibodies, peptides and nucleic acids of the invention are formulated into pharmaceutical compositions, which are prepared by conventional pharmaceutical compounding techniques. For example, Remingto
See n's Pharmaceutical Science, 18th Edition (1990, Mack Publishing, Easton, PA). The composition may contain, in addition to one of the active agents, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such substances are non-toxic and will not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, for example, intravenous, oral, intrathecal, intranervous, parenteral.

For oral administration, the compounds can be formulated into solid or liquid preparations, such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. For preparing compositions in oral dosage form, for oral liquid preparations (eg, suspensions, elixirs and solutions), for example, water, ethylene glycol, oils, alcohols, flavoring agents, storage Any of the usual pharmaceutical media such as agents, colorants, suspending agents, etc .; (for example, powders, capsules and tablets)
Carriers such as starch, sugars, diluents, granulating agents, lubricants, binders, disintegrants and the like may be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to stabilize it, allowing it to pass through the gastrointestinal tract and at the same time cross the blood-brain barrier. See, for example, WO 96/11698.

For parenteral administration, the compounds may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Examples of suitable carriers are water, saline, glucose solutions, fructose solutions, ethanol, or oils of animal, vegetable or synthetic origin. In addition, the carrier can contain other components such as a preservative, a suspending agent, a stabilizer, a buffer and the like. If the compounds are administered intrathecally, they can be dissolved in cerebrospinal fluid.

[0153] The active agent is preferably administered in a therapeutically effective amount. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g., determination of dosage, timing, etc., is within the responsibility of the practitioner or specialist and typically involves the condition to be treated, the condition of the individual patient,
Taking into account the site of delivery, the mode of administration and other factors known to the physician. Examples of procedures and protocols can be found in Remington's Pharmaceutical Science.

Alternatively, targeting therapies can be used to more clearly deliver active agents to certain types of cells through the use of targeting systems such as antibodies or cell-specific ligands. Targeting is desirable for a variety of reasons, for example, if the drug is unacceptable toxicity or if it requires too high a dose or cannot enter the target cells.

Instead of administering these agents directly, they are, for example,
US Pat. No. 5,55,55 designed in a vector or for implantation in a patient
No. 0,050, PCT Application Nos. WO92 / 19195, WO94 / 2
5503, WO95 / 01203, WO95 / 05452, WO96 / 0228
6, can be produced in target cells in a cell-based delivery system such as published in WO 96/02646, WO 96/40871, WO 96/40959, and WO 97/12635. The vector can be targeted to the particular cell to be treated and can contain regulatory elements, tissues that are more specific to the target cell. Cell-based delivery systems are designed to be implanted in a patient's body at a desired target site and contain a coding sequence for the active agent. Alternatively, the drug is
Depending on the active agent produced in or targeted to the cell to be treated, it can be administered in a precursor form for conversion to the active form. See, for example, EP 425,731A and WO 90/07936.

A method for preventing LQT and torsades de pointes. There are various routes for LQT to occur. Specific genes, such as HERG
Mutations can cause LQT. Also treatment with any of the various drugs
Can also cause LQT. These drugs are taken to treat cardiac arrhythmias
What to do, and some such as antihistamines and lithromycin
Also includes other drugs, including antibiotics. LQT is mutated (genetic or familial L
QT) or drug-induced (acquired LQT)
It is due to the effect on ion channels. The drug has its major subunits
Is the K encoded by HERG⁺Channel I_KrInteracts with
Therefore, the heart cell K⁺Affects the flow. Also, mutations in HERG are:
K through this channel⁺Can affect the flow. This results in long-term QT syndrome
Could lead to Torsades de Pointes. Extracellular K⁺Rise in HER
It was found to cause an increase in G current. This is extracellular K⁺Increase outward
K⁺Reduces the chemical driving force for the flow of the flow, and thus increases the outward current
Outly weird effects, because they would be expected to be reduced rather than let
It is. This observation indicates that extracellular K⁺Increase is K⁺Indicates that the channel is activated
. This active compound may be used as a result of mutation in HERG or as a result of drug treatment.
LQT that can result from at least partial inactivation of the channel can be prevented.
You. Normal extracellular physiological K measured in human serum⁺The concentration is about 3.5-4.
In the range of 5 mM. Values in the range of 3-5 mM are frequently found, 2-3 or 5
Few values in the -7 mM range are seen. Sometimes less than 2 mM or more than 7 mM
Can be seen. 5 mM extracellular K⁺HERG current at a concentration of 2 mM
It was found to be 40% greater than the applied current. Extracellular K⁺By increasing the level
K⁺Channel enhancement is beneficial. During a rapid heart rate or ischemia, K⁺Is
Accumulates in the intracellular space. Extracellular K⁺Increases the external current, which
To reduce intracellular accumulation. People with a genetic form of LQT or acquired
Extracellular K in individuals undergoing drug treatment that can cause LQT⁺Level monitoring
Physicians report that the doctor⁺K to the patient with the level ⁺ Would be possible. At least 3.5-4.5m of normal level
M, preferably from said normal level to 4.5-5.5 mM, most preferably
About 5 mM K⁺Up to extracellular K⁺By increasing the level, LQT and
And / or the occurrence of torsades de pointes will be inhibited. Causes of LQT
This new knowledge of patients has increased the risk of developing genetic or acquired LQT for patients.
And extracellular K⁺Monitor the level and check for low or normal extracellular K⁺Have a level
K⁺Will be directed to a system for administering. Such treatments include LQT and / or
Or lead to the prevention of Torsades de Pointes.

In theory, mutations in the cardiac sodium channel gene can cause LQT. Voltage-gated sodium channels mediate rapid depolarization in ventricular myocytes and also lead to small currents during the plateau phase of the action potential (Attwell
1979). Subtle abnormalities in sodium channel function (eg, delayed sodium channel inactivation or altered voltage dependence with channel inactivation)
Delays cardiac repolarization, which can lead to QT prolongation and arrhythmias. In 1992, Gellens and coworkers cloned and characterized the cardiac sodium channel gene, SCN5A (Gellens et al., 1992). The structure of this gene was similar to others previously characterized as a sodium channel encoding a large protein of 2016 amino acids. These channel proteins contain four homology domains (DI-DIV), each of which contains six putative membrane spanning segments (S1-S6). SCN5
A maps to chromosome 3p21, making it a good candidate gene for LQT3 (George et al., 1995), and a late mutation in SCN5A
(Wang et al., 1995).

A mutation in HERG, the cardiac potassium channel gene, results in a genetically linked chromosome 7 linked form of LQT (details are provided in the Examples). The mutations identified in HERG, and the biophysics of the sodium channel alpha subunit, suggest that genetic LQT linked to chromosome 7 may be due to the dominant negative mutations and the reduction obtained in functional channels. Suggests that

[0159] Presymptomatic diagnosis of LQT has relied on the identification of QT prolongation on electrocardiograms. Unfortunately, electrocardiograms are rarely performed in young, healthy individuals. Further, many LQT gene carriers have relatively normal QT intervals, and the first sign of disease may be fatal cardiac arrhythmias (Vincent et al., 1992). Four LQs
Now that the T gene has been identified, genetic testing for this disease is contemplated. This will require continued mutation analysis and identification of additional LQT genes. More detailed phenotypic analysis can reveal phenotypic differences between various forms of LQT. These differences can be useful for diagnosis and treatment.

The identification of the association between HERG, KVLQT1, SCN5A and KCNE1 gene mutations and hereditary LQT was based on early presymptomatic screening of individuals by L
It allows them to identify the risk of developing QT. To identify such individuals, alleles are screened for mutations either directly or after cloning the allele. The allele is tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded conformational analysis (SSCP), linkage analysis, R
Nase protection analysis, allele specific oligonucleotide (ASO), dot
Plot analysis and PCR-SSCP analysis. For example, (1) determine the nucleotide sequence or the appropriate fragment (coding or genomic sequence) of both the cloned allele and the normal HERG gene and then compare them;
The RNA transcript of the ERG gene or gene fragment is hybridized with single-stranded total genomic DNA from the individual to be tested, and the resulting heteroduplex is treated with ribonuclease A (RNase A) and denatured. Run on a gel to detect any mismatches. Two of these methods can be performed according to the following procedure.

Alleles of the HERG gene in the individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from an individual. Genomic DNA isolated from cells in this sample is partially digested to an average fragment size of about 20 kb. Fragments ranging from 18-21 kb are isolated. The obtained fragment is
It is ligated to an appropriate vector. The clones were then sequenced and normal HE
Compared to the RG gene.

[0162] Alternatively, the polymerase chain reaction (PCR) is performed using primer pairs for the 5 'region or exons of the HERG gene. In addition, PCR may be performed using primers based on any sequence of a normal HERG gene.
For example, a primer pair of one of the introns can be prepared and utilized. Finally, RT-PCR may also be performed on the mRNA. Amplification products were analyzed by single-stranded conformational analysis (SSC) using conventional techniques to distinguish any differences.
P) to identify any differences, which are then sequenced and compared to the normal gene sequence.

Amplification of individual DNA using the use of appropriate primer pairs and analysis of amplification products, for example, by common HERG gene mutation by dot blot using an allele-specific oligonucleotide probe The body can be screened quickly.

The second method uses RNase A to help detect differences between the normal HERG gene and the defective gene. The comparison is made in a step using a small (about 500 bp) restriction fragment of the HERG gene as a probe. First, the HERG gene is digested with restriction enzymes, cutting the gene sequence into fragments of about 500 bp. These fragments are individually separated on an electrophoresis gel, purified from the gel, and cloned in both orientations into an SP6 vector (eg, pSP64 or pSP65). The SP6-based plasmid containing the insertion of the HERG gene fragment was [α- ³² P
] Transcribed in vitro using the SP6 transcription system well known in the art in the presence of GTP to obtain radiolabeled RNA transcripts of both strands of the gene.

Individually, using these RNA transcripts, allelic DNA was synthesized using conventional techniques.
To form a heteroduplex. The mismatch caused in the RNA: DNA heteroduplex due to sequence differences between the HERG fragment from the individual and the HERG allele subclone results in cleavage of the RNA strand when treated with RNaseA. Such mismatches may be the result of a point mutation or small deletion in an individual's allele. Cleavage of the RNA strand results in two or more small RNA fragments,
Runs faster on denaturing gels than the A probe itself.

[0166] Any differences found will identify molecular variants of the HERG gene, as well as individuals with an inevitable presence of LQT. These variants can take many forms. Most severe forms are frameshift mutations or large deletions that cause the gene to code for an abnormal protein, or that significantly alter protein expression. Less severe destructive mutations include small in-frame deletions and non-conservative base pair substitutions, including changes to or from cysteine residues, changes from basic to acidic amino acids, and vice versa. Such changes from hydrophobic to hydrophilic amino acids or vice versa, or other mutations that affect secondary or tertiary protein structure, will have a significant effect on the protein produced. Silent mutations or those that produce conservative amino acid substitutions were generally not expected to disrupt protein function.

Genetic testing allows physicians at birth or before birth to identify individuals at risk for genetic LQT. Presymptomatic diagnosis of LQT will allow prevention of these diseases. Existing medical treatments, including beta-adrenergic blockers, can prevent and delay the onset of disease-related problems. Finally, the present invention changes the inventors' understanding of the causes and treatment of common heart diseases such as cardiac tachyarrhythmias, which account for 11% of all spontaneous deaths. Existing diagnoses have focused on measuring QT intervals from electrocardiograms. This method is not a sufficiently accurate indicator of the presence of long-term QT syndrome. The present invention is a more accurate indicator of the presence of a disease.

Association between HERG and acquired LQT HERG encodes a K ⁺ channel with inward rectification properties similar to I _Kr . To determine the physiological properties of HERG, the full-length cDNA was cloned,
Characterized. It was prepared for expression in Xenopus oocytes. The characteristics of the expressed channel were determined using standard 2-microelectrode voltage clamp technique using cRN.
Tested on oocytes 2-6 days after A injection. The HERG current is the test potential>
Activated in response to -50 mV. The magnitude of the HERG current is -10 mV (FIG. 1).
A) progressively increases with test potential until A), then progressively> 0 with test potential
mV (FIG. 1B). The deactivation of the current (tail current)
It was examined after the return of the membrane to a holding potential of -70 mV. Amplification of the tail current
It increased gradually after depolarization and was saturated at +10 mV at +. HERG current-voltage (IV) relationships measured for 10 oocytes are shown in FIG. 1C. The peak external current decreased with increasing depolarization, which indicated that HERG was an internal rectifier. The voltage dependence of channel activation was examined by plotting the relative amplification of tail current as a function of test potential (FIG. 1D). HERG is
A half-value activation was reached at a potential of −15.1 mV. These data define HERG as a rectifier K ⁺ channel with almost the same voltage dependence of activation and rectification characteristics as I _Kr (Sanguinetti and Jurkiewicz, 1990; Shibasaki,
1987; Yang et al., 1994). These properties are different from other cardiac currents.

To further characterize HERG, current activation and deactivation over time was measured. The onset (activation) of the current over time fitted best with a single exponential (FIG. 2A). The rate of activation increased with increasing change at test potentials from -40 to +50 mV. Current deactivation, like _IKr , best fit a bi-exponential function (FIG. 2B) (Chinn, 1993; Yang et al., 1994). The time constant for activation of the HERG current, and the fast deactivation phase, was a bell-shaped function of the test potential (FIG. 2C). The relative amplification of the fast deactivating component varied from 0.77 at -30 mV to 0.2 at -120 mV (Figure 2D). The kinetics of the HERG current is slower than I _Kr (Sanguinetti and Jurkiewicz, 199
0; Shibasaki, 1987; Yang et al., 1994), showing the same voltage dependence.

The HERG current is activated by extracellular K ⁺ . The K ⁺ selectivity of HERG was determined by comparing N with different concentrations of KCl (0.5-20 mM).
It was determined by measuring the reverse potential of the current in oocytes placed in D96 solution. Tail current was measured at variable test potentials after activation of the current by pulses up to +20 mV (FIGS. 3A and 3B). The voltage that reversed the tail current from the inside to the outside current was defined as the reverse potential, E _rev . This is because [K ⁺ ] _e > 5 mM
As predicted by the Nernst equation for the extracellular K ⁺ concentration ([
K ⁺ ] _e ) (change of 58 mV for a 10-fold increase in [K ⁺ ] _e ). E _{r ev} is Goldman - Hodgkin - has changed over the entire range of ^[K _{+] e} in well-described method by the current equation of Katz (Figure 3C). These data indicate that HERG is selectively permeable to K ⁺ over Na ⁺ by a factor of 143.

A prominent feature of cardiac I _Kr is its regulation by [K ⁺ ] _e (Sanguinett
i and Jurkiewicz, 1992). The effect of [K ⁺ ] _e on the magnitude of the HERG current is shown in FIGS. 4A-C. HERG currents increased in direct proportion to [K ⁺ ] _e , but the IV-related shape was not altered (FIG. 4D). [K ⁺ ] _e dependence of HERG current was measured by comparing peak outward currents at +20 mV in oocytes in a solution containing 0.5 to 20 mM KCl. Over this range, the current amplification of HERG varied as a linear function of [K ⁺ ] _e (FIG. 4E). Unlike most other K ⁺ currents, the magnitude of the outer HERG current is
It decreases oddly with removal of extracellular K ⁺ .

Rectification of the HERG current results from rapid channel inactivation. The inward rectification of I _Kr is postulated to be due to voltage-dependent inactivation, which is more rapid than activation (Sanguinetti and Jurkiewicz, 1990; Shibasaki, 19
87). The end result of the two competing processes is the steady-state activation variable for the outward K ⁺ flow and the magnitude of the reduced current relative to that expected from the electrochemical driving force. It is assumed that the peak tail current does not show a similar rectification after strong depolarization (see FIG. 1), since the channel recovers from a faster deactivation than the inactivation over time. If this interpretation were correct, it would be possible to measure recovery over time from fast inactivation during the onset of tail current. FIG. 5 shows the results of this experiment. Tail currents were recorded at several test potentials, each at +40 m
Preceded by a pre-pulse to V (FIG. 5A). The voltage dependence of the time constant for recovery from fast inactivation is plotted in FIG. 5B. Recovery is -30mV
(T = 18.6 milliseconds), slower and faster with increasing or decreasing test potential. The bell-shaped association between the time constant for recovery from inactivation and the membrane potential peaked at the same voltage (-30 mV) as the relationship describing the voltage dependence of current activation and deactivation of HERG. (FIG. 2C). Although the onset of fast inactivation cannot be quantified (since it occurs much faster than activation), the descending leg of the curve in FIG. It seems to show. These data indicate that the inward rectification of the HERG current results from an inactivation process that is much faster than activation over time.

The voltage dependence of channel rectification was determined by comparing fully activated IV association for HERG current (FIG. 5C) with the expected IV association for ohmic conduction. The dashed line in FIG. 5C was extrapolated from a linear fit of the current amplification measured at -90 to -120 mV, which showed the IV relationship occurring in the absence of inward rectification (ohmic conduction). The slope of this line defines the maximum conductance of HERG in this oocyte (118 microseconds) and is used to calculate the voltage dependence of channel rectification (FIG. 5D). Rectification was half-valued at -49 mV, and the association had a slope factor of 28 mV. Half of the points were very similar to I _Kr in rabbit nodule cells, and their slope coefficients were almost identical to I _Kr in guinea pig myocytes (Table 2).

The steady state HERG current at any given test potential (V) can be defined as: I _HERG = _GnR (V _t -E _rev ) where: G = HERG current Maximum conductance; n = activation variable; R = rectification variable; E _rev = reverse potential.

The HERG current is blocked by lanthanum and cobalt, but is not affected by methanesulfonanilide or cyclic nucleotide. I _Kr of cardiomyocytes is 10-100 μM lanthanum (La ³⁺ ), 2 mM cobalt (Co ²⁺ ) (Balser et al., 1990; Sanguinetti and Jurkiewicz, 1
990), and some methanesulfonanilide antiarrhythmic drugs at nM concentrations such as E-4031 (Sanguinetti and Jurkiewicz, 1990) or MK-499 (Lynch et al., 1994). It was determined whether the HERG current was also blocked by these cations and drugs. 0mV
At a test potential of 10 μM La ³⁺ reduced the HERG current by 92 ± 3% (
n = 4, FIG. 6). At least part of the La ³⁺ blocking effect is due to the negative membrane surface charge as shown by the 40 mV positive shift in both the IV-related peak (FIG. 6C) and the activation curve at the same time (FIG. 6D). Due to screening (Sanguinetti and Jurkiewicz, 1990). Also, HERG
Was partially blocked by 2 mM Co ²⁺ (n = 2) (52%).
However, also, both E-4031 and MK-499 at a concentration of 1 [mu] M, even after culturing oocytes with these drugs for up to 4 hours.
G current was not blocked. HERG channels contain segments of homology to cyclic nucleotides that bind to a domain near the carboxyl terminus (Warmke and Ganetzky, 1994). To determine if HERG is sensitive to cyclic nucleotides, 8-Br-cAMP and 8-B on expressed HERG currents
The effect of r-cGMP was tested.

[0176]

[Table 2]

These membrane permeable analogs of endogenous cyclic nucleotides have been shown to increase the size of other channels expressed in Xenopus oocytes (Blumenthal and Kaczmarek, 1992; Bruggemann et al., 1993). None of the compounds had a significant effect on the current magnitude or voltage dependence of channel activation at a concentration of 1 mM within 30 minutes of application (data not shown).

HERG is the heart I_KrCode the subunit of The above results indicate that HERG is_KrCoding the main subunits of the channel
Indicates that HERG expressed in oocytes is associated with I in cardiomyocytes. _Kr (Table 2) that induce currents that share most of the distinguishing characteristics that define This
These include: 1) IV-related inward rectification with a peak near 0 mV; 2)
Voltage dependence of activation; 3) [K⁺]_eStrange regulation of the current by the current; and 4) La ³⁺ Or Co²⁺Block by. Activation Kinetics and HERG
Inactivation of the current was measured at room temperature by measuring the I of mouse AT-1 cells._KrMuch more
Late (Yang et al., 1994). This difference may be due to some intrinsic factors or additional factors.
The channel subunit is the heart cell I_KrAdjust channel gating
It may indicate that. In addition, HERG is controlled by 8-Br-cAMP.
Isoproterenol does not activate I_KrDo not increase
(Sanguinetti et al., 1991). Therefore, in the oocyte
The co-assembly of the HERG subunit is probably a homotetramer
) As the heart I_KrCan reconstruct key biophysical properties of MacKinnon
1991). Other channels may not share all these characteristics.

HERG current and I_KrThe only major difference between
I_KrA powerful and specific blocker of methanesulfonanilide drug (E-4)
031, MK-499) does not block HERG
(Lynch et al., 1994; Sanguinetti and Jurkiewicz, 1990). This is _Kr Channels and methanesulfonanilide receptors are separate but interact
Suggest that this is a protein. A similar phenomenon has recently been isolated from mammalian hearts.
K_ATPChannels have been described (Ashford et al., 1994). this
Channel (rcK_ATPIf -1) is expressed in HEK293 cells,
Is the biophysical properties of natural channels, including regulation by intracellular nucleotides
(Ashford et al., 1994). However, the channel
, K in cardiomyocytes_ATPGlibenclamide, a drug that inhibits
Not locked (Ashford et al., 1994). Dofetilide or
Using known high affinity probes such as MK-499 biochemically,
It may be possible to isolate the honanilide receptor. Methanesulfoneanily
Co-expression of HERG channels with a dopamine receptor is responsible for the interaction between these two molecules
Will allow for detailed testing.

The mechanism of HERG rectification is rapid channel inactivation. A unique feature of _IKr is IV-related inward rectification. Inner rectifier of the heart,
I _Kr exhibits stronger inward rectification, which occurs beyond the more negative voltage range. At under normal physiological conditions, _{I KI} of the outer peak occurs at -60 mV, _{whereas, I Kr} is the peak at 0 mV. The mechanism of I _KI commutation, ^{Mg 2+} intracellular (Vandenberg, 1987) and spermine (Fakler et al., 1995) due to both a voltage-dependent gating mechanism and the outer current block by.
In contrast, it was assumed to be due to the voltage dependence of inactivation its inward rectification of I _Kr rectification occurs much faster than activation (Shibasaki, 1987). The kinetics of fast inactivation is difficult to analyze in recording macroscopic currents in myocytes, and was therefore calculated based on the kinetics of single channel activity (Shibasaki, 1987). In this test, it is possible to analyze the time course recovery from macroscopic HERG current inactivation due to the large signal-to-noise ratio at room temperature and the relatively slow channel gating kinetics. Met. The rapid onset and recovery form of fast inactivation explains the significant IV-related inward rectification for HERG. For example, at a test potential of +20 mV, HERG is activated with a time constant of 230 ms, but is simultaneously deactivated with a time constant of 12 ms. Thus, the activation of the current is completed before reaching a significant level, so that the amplification of the current is greatly reduced. Recovery from inactivation
It occurs quickly for depolarization and its tail current amplification does not significantly affect it after repolarization. The inventors have found that the mechanism of rectification for I _Kr (and HERG) is
We support Shibasalci's hypothesis that it is a rapid voltage-dependent inactivation.

HERG current rectification, half value (V _1/2 ) at -49 mV, with a slope factor of 28 mV. The slope coefficient of HERG rectification was similar to I _Kr measured in guinea pig myocytes (22 mV). V _1/2 of HERG rectification is equal to guinea pig (
More negative than estimated in Table 2). However, the voltage dependence of I _Kr rectification in guinea pig myocytes was difficult to measure due to overlap with very large I _KI at negative test potentials. The lack of overlapping electrical currents in rabbit nodule cells and oocytes expressing HERG,
These determinations were similar, allowing a more accurate measurement of the voltage dependence of the channel rectification (Table 2). Single channel analysis of the expressed HERG will allow a more detailed description of voltage-dependent gating and fast inactivation.

[K of the HERG current⁺]_eDependency can regulate the duration of the cardiac action potential. [K⁺]_eIncrease caused an increase in outward HERG current. [K⁺]_eIncrease
K is outside K⁺Reduce the chemical driving force for the flow of
This is odd because it is expected to decrease rather than increase the current
The result. The same phenomenon is I_KrHas been described (Sanguinetti and
And Jurkiewicz (1992); Samps and Carmeliet, 1989), I._KIOther than
No other cardiac channels are described. However, I _KI Is almost immediately activated by hyperpolarization, but HERG_KrLike
It is activated relatively slowly by polarization and is not activated by hyperpolarization.

Modulation of HERG (and I _Kr ) by [K ⁺ ] _e may have physiological significance. During rapid heart rate or ischemia, K ⁺ accumulates in the intracellular space (Gintant et al.,
1992). This increase in [K ⁺ ] _e is due to the HER to net repolarization current.
G (I _Kr ) will increase the contribution. Therefore, HERG (I _Kr ) is
It is even more important in regulating the action potential duration of high heart rates or during the early phases of ischemia.

The mechanism of HERG regulation by [K ⁺ ] _e is not yet known, but may be similar to that described for the other cloned K ⁺ channel, RCK4.
Also, RCK4 amplification increases with increasing [K ⁺ ] _e (Pardo et al., 199).
2). Single channel analysis revealed that an increase in [K ⁺ ] _e increased the number of channels that could open, but had no effect on single channel conductivity, average opening time or gating time ( Pardo et al., 1992). In addition, substitution of a single lysine located near the channel pore with a tyrosine residue (K533Y)
It was demonstrated that this effect was eliminated. A similar [K ⁺ ] _e- dependent increase in current was caused by substitution of a single amino acid near the pore region of the Shaker B channel (Lopez-Barneo et al., 1993). Future experiments will determine whether K ⁺ regulates a single HERG channel by a similar mechanism.

[0185] HERG mutations, and _{I Kr} of drug-induced block: mechanistic linkage between genetic and acquired LQT. Genetic LQT and the more common (drug-induced) acquired forms of the disorder are:
Torsades de Pointes, associated with polymorphic ventricular tachyarrhythmia. Mutations in HERG have recently been shown to cause chromosome 7 linked LQT, such as by a dominant negative inhibition of HERG function (Curran et al., 1995). It should be noted that there may be several different mechanisms that explain acquired and genetic LQT. For example, it has recently been demonstrated that mutations in SCN5A, a cardiac sodium channel gene, cause a chromosome 3 linked LQT (
Wang et al., 1995). Discovery that HERG to form I _Kr channel is logical for the observation that block I _Kr by certain drugs can induce the same arrhythmia to that observed (torsade de Powantsu) in familial LQT Provide an explanation.

This finding may have important clinical implications. Physiological range [K
⁺]_eChanges significantly regulated the amplification of the HERG current. For example,
[K from a level of 2 mM to a new level of 5 mM⁺]_eRise in HERG
The current was increased to 40%. Mild hypokalemia of a common clinical problem is
, Will have a significant effect on the HERG current. This is hypokalemia
Explain the link between disease and acquired LQT (Roden, 1988). Furthermore, low power
Liumemia itself has been associated with ventricular arrhythmias (Curry et al., 1976). Those
Moderately prolongs the action potential of the heart, thereby causing the re-entra
nt) As it inhibits arrhythmias, I_KrDrug treatment to reduce (eg sotalol)
(Sotalol), dofetilide) can be effective antiarrhythmic agents. However,
In hypokalemia situations, this effect is exaggerated, resulting in excessive action potential prolongation and
Will lead to the guidance of Russard de Points. These antiarrhythmic drug treatments
Patients who have received or who have received other drugs that may cause acquired LQT
[K] in normal subjects and individuals with chromosome 7 linked LQT⁺] Reasonable
Rise will help prevent LQT and Torsades de Pointes
.

In summary, it has been demonstrated that HERG encodes the major subunit that forms the I _Kr channel. The findings suggest that the molecular mechanism of LQT linked to chromosome 7 and certain acquired forms of the disease may be due to dysfunction of the same ion channel.

The present invention is further described in the following examples, which are provided by way of illustration and are not intended to limit the invention in any way. Standard techniques well known in the art or techniques described in detail below are utilized.

Example 1 Method of Assessing Phenotype LQT relatives were identified from medical clinics throughout North America. The phenotypic criteria are:
Conventional testing (Keating et al., 1991a; Keating et al., 1991b; Keating, 19
92). Individuals have a heart rate (QTc; newspaper, 1
LQT based on QT intervals modified per 920) and the presence of unconsciousness, seizures and abnormal sudden death. The consent provided was obtained from each individual or their guardian in accordance with the local institutioonal review boad guideline. Phenotypic data was interpreted without knowledge of the genotype. As affected, they were assigned to symptomatic individuals with a modified QT interval (QTc) of 0.45 seconds or more and asymptomatic individuals with a QTc of 0.47 seconds or more. . 0.4 as unassignable
Asymptomatic individuals with a QTc of 1 second or less were classified. As ambiguous, asymptomatic individuals with a QTc between 0.41 and 0.47 seconds and symptomatic individuals with a QTc of less than 0.44 seconds were classified.

Example 2 Linkage Analysis Linkage analysis of pairs was performed using MLINK in LINKAGE v5.1 (La
throp et al., 1985). 0.90 per osmosis and 0.9 per LQT gene frequency.
An estimate of 001 was used. Gene frequencies were assumed to be equal between males and females.

Example 3 Isolation of HERG Genomic and cDNA Clones The HERG probe was prepared using human genomic DNA and primer pairs 1-10,6.
Were generated using the products of the PCR reaction using -13 and 15-17 (Table 3).
These products were cloned and radiolabeled to high specific activity and used to screen a human genomic P1 library (Sternberg, 1990).
. Positive clones were purified, characterized, and used for FISH and DNA sequence analysis. A HERG genomic clone containing the domains S1-S3 and intron I (Curran et al., 1995) (where intron 6) was used to construct a human hippocampus cDNA library (Stratagene, library # 936205) with approximately 1 part.
0 were screened ^six recombinants. Nucleotide 3 of the HERG coding sequence
A single, partially processed cDNA clone containing 2-2398 was identified. A second screening of this library was performed using the coding portion of this cDNA. This screen generated a second clone containing the HERG coding sequence from nucleotide 1216 through the 3 'untranslated region (UTR) and containing a poly A ⁺ region. These two cDNAs were ligated using an XhoI site at position 2089. To recover the 5 'region of the HERG, about ¹⁰⁶ clones of a human heart cDNA library (Stratagene, library # 936207) was screened with cDNA composite hippocampus. A single clone containing the 5'-UTR through nucleotide 2133 was isolated. This clone was ligated to the hippocampus complexed at the BgIII site (nucleotide 1913) to obtain the full-length HERG cDNA.

Example 4 YAC-based mapping of HERG Using the 3 ′ untranslated region of HERG (primers 5 ′ GCTGGGCCGCTCCCCTTGGA 3 ′ (SEQ ID NO: 7) and 5 ′ GCATCTTCATTAATTATTCA 3 ′ (SEQ ID NO: 8), 309
A collection of YAC clones highly enriched for human chromosome 7 was screened using a PCR assay specific for the bp product (Green et al.
Printing). Two positive YAC clones were identified (yWSS2193 and yWSS1759), both of which were identified by the genetic marker D7S505 (Green et al., 19).
94) were included in a large contig containing a positive YAC.

Example 5 Fluorescent in Situ Hybridization Metaphase chromosome spreads were prepared from normally cultured lymphocytes (46X, Y) by standard procedures of colcemide arrest, treatment of hypotonicity and acetic acid-methanol fixation. HERG P1 clone 16B4 was prepared from biotin-14 by standard methods.
Labeled by incorporation of dATP (BioNick system, Gibco-BRL), hybridized to metaphase spreads and detected with streptavidin-Cy3 (Lichter et al., 1988). To identify chromosome 7, α-satellite probes (Oncor) specific for digoxigenin-labeled centromeres were hybridized together and detected with anti-digoxigenin-FITC. Chromosomes were counterstained with DAPI and visualized directly on a micrograph.

Example 6 SSCP Analysis Genomic DNA samples were amplified by PCR and used for SSCP analysis as described (Orita et al., 1989; Ptacek et al., 1991). Table 3 shows the primer pairs used for this test. Annealing temperature was 58 ° C for all PCR reactions
Met. The reaction (10 μl) was diluted with 40 μl of 0.1% SDS / 1 mM EDTA and 30 μl of 95% formamide dye. The diluted product was denatured by heating at 94 ° C. or 100 ° C. for 5 or 10 minutes, and 3
-5 μl was separated by electrophoresis at 4 ° C. on either 7.5% or 10% native polyacrylamide gel (50 acrylamide: 1 bis-acrylamide). Electrophoresis was performed at 40-50 watts for 2-5 hours. The gel was transferred to 3MM filter paper, dried and exposed to X-ray film at -80 ° C for 12-36 hours.

[0195]

[Table 3]

Example 7 Sequence analysis of the SSCP conformer. Normal and abnormal forms of the SSCP conformer were cut directly from the dried gel and eluted in 100 μl of distilled water at either 37 ° C. or 65 ° C. for 30 minutes.
10 μl of the eluted DNA was used as a template for a second PCR reaction using the original primer pair. The products were fractionated in a 2% low melting temperature agarose gel (FMC), the DNA fragments were purified and sequenced directly by cyclic sequencing (Wang and Keating, 1994). Or,
The purified PCR product was cloned into pBluescript II SK ⁺ (Stratagene) using a T-vector as described (Marchuck et al., 1990). Plasmid DNA samples were purified and purified by dideoxy.
Sequence was determined by the chain termination method.

Example 8 Exon / Intron Bounding Screening of a human cosmid library spans approximately 55 kb.
Two cosmids covering all exons were given (FIG. 7). All genomic clones were sequenced using primers designed for cDNA sequencing. HERG cosmids were sequenced by the dideoxy chain termination method on an Applied Biosystems model 373A DNA sequencer. The exact exon / intron boundaries were determined by comparison of the cDNA, genomic sequence and known price site consensus sequence.

Exon / intron boundaries were determined by sequencing cosmids with primers designed for the cDNA. Sequencing is performed at 100 bp (
15 with a size ranging from exon 11) to 553 bp (exon 15)
Exons (FIG. 8) were revealed (see Table 4). Intron donor and acceptor splice sites did not deviate from uniform GT and AG. A single pair of primers was designed for most exons, and two pairs with overlapping products were designed for exons 4, 6 and 7 (Table 5). Exons 1 and 11 were amplified using nested PCR with repetitive DNA sequences flanking the intron. This set of primers can be used to screen the entire coding sequence of HERG for mutation.

[0199]

[Table 4]

[0200]

[Table 5]

Example 9 Design of PCR Primers and PCR Reaction Conditions Primers for amplifying the exons of the HERG gene were experimentally or OL
Designed using IGO 4.0 (NBI). The amplification conditions were: (1) 94 ° C. for 3 minutes, followed by 94 ° C. for 10 seconds, 58 ° C. for 20 seconds, and 72 ° C.
30 cycles of 20 seconds, and 5 minutes extension at 72 ° C. (2) Same conditions as (1), but the reaction had a final concentration of 10% glycerol and 4% formamide and was covered with mineral oil. (3) 94 ° C. for 3 minutes, followed by 94 ° C. for 10 seconds, 64 ° C. for 20 seconds, and 72 ° C.
5 cycles of 20 seconds and 94 ° C for 10 seconds, 62 ° C for 20 seconds and 72 ° C2
30 cycles of 0 seconds and extension at 72 ° C for 5 minutes. In a nested PCR of exons 1 and 11 of HERG, a 2 μL aliquot from the initial reaction was used for the second reaction.

Example 10 Northern Analysis A multiple tissue Northern blot containing 〜2 μg / lane of poly-A ⁺ mRNA was obtained from Clontech (Human MTN blot 1). High specific activity (> 1.5 × 10 ⁹ cpm / μg) containing nucleotides 679-2239 of the coding sequence
DNA), by the described random hexamer initiation [Feinberg and Vogelstein,
1983], a cDNA fragment of radiolabeled HERG was prepared. Probes were added to the hybridization solution at a final concentration of 5 × 10 ⁶ cpm / ml. Hybridization was performed at 42 ° C. for 24 hours in 20 ml Quickhyb (Stratagene). Final wash at 65 ° C. in a solution of 0.1% SDS / 0.1 × SSC
For 30 minutes.

Example 11 Linkage Analysis of HERG LQT2 was linked to a marker on chromosome 7q35-36 with three known L
To determine the relative frequency of the QT loci (LQT1, LQT2, LQT3)
Linkage analysis was performed in families with this disease. Five LQT families were identified and characterized by genotype (FIG. 13). These families are irrelevant, have different families,
Included Mexicans (Spanish), Germans, British and Danes. In each case, an autosomal dominant inheritance pattern was suggested by pedigree survey. Affected individuals were confirmed by the presence of a prolonged QT on the electrocardiogram and, in some cases, by a history of syncope or aborted sudden death. All patients have signs of congenital neural hearing loss, a rare autosomal recessive form of LQT
Had no findings, or other genotypic abnormalities. Genotyping with polymorphic markers linked to the known LQT locus indicated that the disease genotype in these families was linked to the polymorphic markers on chromosomes 7q35-36 (FIG. 1).
4). Maximum combinatory two-point rod score (maximum combin) for these five families
ed two-lod score) was 5.13 in D7S636 (θ = 0.0; Table 6)
. Combined with previous studies [Jiang et al., 1994; Wang et al., 1995], the maximum combined two-point rod score for the 14 chromosome 7-joined families was similarly D7S636.
Was 26.14 (θ = 0.0; Table 6). Haplotype analysis was consistent with previous studies, placing LQT2 between D7S505 and D7S483 (Figure 14; Wa
ng et al., 1995), localized this gene to chromosome 7q35-36.

HERG Map for Chromosome 7q35-36 HERG was previously mapped to chromosome 7 [Warmke and Ganetzky, 1994]. To test the candidate nature of this gene, HERG localization was improved using two physical mapping techniques. First, HERG was constructed against a set of yeast artificial chromosomes constructed against chromosome 7.
YAC) mapped on the contig [Green et al., 1994]. HERG is LQ
It was localized to the same YAC as D7S505, a polymorphic marker that strongly bound to T2 (Table 6). Second, using fluorescence in situ hybridization (FISH) with a P1 genomic clone containing HERG,
G was mapped to chromosomes 7q35-36.

To determine whether HERG is genetically linked to the LQT locus, polymorphisms in HERG were confirmed using SSCP analysis and linkage analysis was performed on the chromosome 7-
Performed in a combined family. Two abnormal SSCP conformers were identified in DNA samples from patients and controls using primer pairs 5-11 and 3-8. Three conformers were cloned and sequenced. One aberrant conformer resulted in a C to T substitution at position 3 of codon 489 (DNA nucleotide 1467, observed heterozygosity = 0.37). Another abnormal conformer is codon 564 (cDNA nucleotide 1692, observed heterozygosity = 0.44
) Resulted in an A to G substitution at position 3. None of the substitutions affected the HERG predicted amino acid sequence. HERG polymorphism was used for genotyping in chromosome 7-linked families (FIG. 9). No recombination events were identified between HERG and LQT in any of these families. The maximum combined rod score for the 14 families was 9.64 (θ = 0.0; Table 6). These data indicate that HERG is completely bound to LQT2.

[0206]

[Table 6]

HEQ gene deletion associated with LQT in two families. HERG is LQT
To test the hypothesis of 2, SSCP analysis was used to screen for mutations in affected individuals. Because the genetic structure of HERG is unknown (this part of the study was performed before determining the complete intron / exon structure of the gene), oligonucleotide primer pairs were published [Warmke and Ganetzk
y, 1994] designed from the HERG cDNA sequence (Table 3). In most cases,
A single product of the expected size was produced. However, primer pairs 1-1
For 0, 6-13, and 15-17, products larger than the expected size were obtained, indicating the presence of intron sequences. To test this possibility, these larger products were cloned and sequenced. DNA sequence analysis was performed using the cDNA sequence SEQ ID NO: 1557/1558, 1945/1946, and 2398/2.
At 399, three introns were identified (FIG. 15). These boundaries were confirmed by direct DNA sequencing of HERG-containing HERG genomic clones (data not shown). Additional primers were designed for intron sequences to facilitate SSCP analysis.

As indicated previously, SSCP analysis using primer pairs 3-8 was performed using HERG
Within, a polymorphism from A to G was confirmed (cDNA nucleotide 1692). This S
Analysis of kin 2287 (K2287) using SCP polymorphisms defined a genotype pattern consistent with null alleles (FIG. 13). Possible explanations for these findings include multiple misinheritances, the possibilities of which are not supported by previous genotyping, DNA sample errors, base pair substitutions or deletions. To test the hypothesis that the genotype data was due to a small deletion, K2287
PCR analysis was repeated with new primer pairs (3-9) flanking the previous set of primers. These experiments confirmed two products of 170 bp and 143 bp in affected members of K2287 (FIGS. 10A and 10B). In contrast, only a single product of 170 bp was observed in unaffected members of this relative. In addition, only a single product of 170 kb was found in DNA samples from more than 200 unaffected individuals. The 143 bp and 170 bp products were cloned from affected individual II-2. The abnormal PCR
Direct sequence analysis of the product revealed a 27 bp deletion starting at position 1498 (ΔI500-F50
8) was clarified. This deletion disrupts the third membrane spanning domain (S3) of HERG.

To further examine the hypothesis that HERG is LQT2, additional SSCPs
The analysis was performed on additional relatives. SSC using the primer pairs 1 to 9
P-analysis confirmed abnormal conformers in K2595 affected individuals (FIG. 11A). Analysis of more than 200 unaffected individuals did not show this exception. The normal and abnormal conformers were cloned and sequenced to reveal a single base deletion at position 1261 (Δ1261). This deletion results in a frameshift in the sequence encoding the first membrane spanning domain (S1), resulting in a new stop codon of up to 12 amino acids (FIG. 11B). Confirmation of the HERG intragenic deletion in the two LQT families indicates that HERG mutations can cause LQT.

Seven HERG point mutations associated with LQT. To confirm additional HERG mutations, further SSCP analysis was performed in relevant relatives and sporadic cases. Three abnormal conformers were identified in affected members of K1956, K2596 and K2015 (FIGS. 12A, 12C and 12D) and five other relatives (K1663, K2548, K2554, K1697 and K1789).
Also showed abnormal bands (FIGS. 13A-E). In each case, the normal and abnormal conformers were cloned and sequenced. In K1956, the C at position 1682 (with the starting codon starting at base 1 for all data in this paragraph)
Was replaced with T. This mutation results in a highly conserved substitution of valine for alanine at codon 561 (A561V), altering the fifth membrane spanning domain (S5) of the HERG protein (FIG. 12B). K259
In 6, the substitution of A to G was confirmed at position 1408. This mutation results in a highly conserved substitution of asparagine for aspartic acid at codon 470 (N470D) and is located in the second membrane spanning domain (S2; FIG. 12D). In K2015, substitution of G for C was confirmed. This substitution disrupts the splice-donor sequence of intron III (intron 9) and affects the cyclic nucleotide binding domain (FIG. 12F). K1663 has a G1714T mutation that results in G572C, and K2548 has an A1762G that results in N588D.
With mutations, both K2254 and K1697 have C
Has the 1841T mutation, and K1789 is a T1889 that gives rise to V630A.
It had a C mutation. No abnormal conformers were identified in DNA samples from more than 200 unaffected individuals. In line with the above studies, further studies revealed some additional mutations in HERG, which were found in patients diagnosed with LQT, but not in 200 unaffected people. These additional mutations are shown in Table 7.

[0211]

[Table 7]

[0212]

[Table 8]

[0213]

[Table 9]

Novel HERG mutations in sporadic cases of LQT. To demonstrate that the HERG mutation results in LQT, we screened for mutations in sporadic cases using SSCP analysis. Primer pair 4-12 identified an abnormal conformer in individual II-1 affected by K2269 (FIG. 14A). This conformer was not identified in any of the parents or in more than 200 unaffected individuals. Direct DNA sequencing of the abnormal conformer was 18
At position 82, substitution of G for A was confirmed. This mutation results in a substitution of serine for glycine, which is highly conserved at codon 628 (FIG. 14B), altering the pore-forming domain. Genotyping of this relative using nine informative STR polymorphisms confirmed maternal and paternal lines. Confirmation of a novel mutation in sporadic cases has established that HERG is LQT2. K1697 and K
A new mutation at 1789 also occurred. Maternal and paternal lines were confirmed in both cases using highly polymorphic short tandem repeats (data not shown).

HERG is expressed in the heart. HERG was originally identified from a hippocampal cDNA library [Warmke and Ganetzky, 1994]. To determine the tissue distribution of HERG mRNA, a partial cDNA clone was isolated and used for Northern analysis. Northern analysis showed the strongest hybridization to cardiac mRNA with weak signals in brain, liver and pancreas (FIG. 15). Non-specific hybridization was also seen in the lung, probably due to genomic DNA contamination. The size of the band observed in cardiac mRNA was consistent with the expected size of HERG. Two bands of ~ 4.1 and 4.4 kb were identified, probably due to alternative splicing or the presence of another mRNA of interest. These data indicate that HERG is strongly expressed in the heart, consistent with its involvement in LQT.

[0216] Mutations in HERG are one cause of LQT. It can be concluded that the mutation in HERG causes a chromosome 7-linked form of LQT. Several lines of evidence support this conclusion. First, using linkage analysis, LQ in 14 families
The T locus (LQT2) was mapped to chromosome 7q35-36. Second, HERG was placed on the same chromosomal region as LQT2 using physical and genetic mapping. Third, HERG has been demonstrated to be expressed in the heart. Fourth, in two families, an intragenic HERG deletion for LQT was identified. Fifth, four HERG point mutations were identified in LQT patients. Finally, 3
One point mutation newly occurred in a highly conserved region encoding a potassium-selective pore domain.

The data suggest a more likely molecular mechanism for chromosome 7-linked LQT. Although the function of HERG was unknown, analysis of its predicted amino acid sequence indicates that it encodes the potassium channel α-subunit. Potassium channels are formed from four α-subunits [MacKinnon, 1991],
It is either a homo- or heterotetramer [Covarrubias et al., 1991]. These biophysical observations suggest that a combination of normal and mutant HERGα-subunits could form abnormal HERG channels. This raises the possibility that the HERG mutation may have a dominant negative effect on um channel function.

The identified mutations are consistent with a dominant negative mechanism. Two mutations result in a premature stop codon and a excised protein (Δ1261 and splice-donor mutation). In the first case, the amino terminus and the first membrane spanning domain (
Only the part of S1) remains. In the second case, the carboxyl terminus of the protein is excised, leaving all membrane spanning domains intact. HERG contains a cyclic nucleotide binding domain near the carboxyl terminus, and in both mutations this domain is deleted. In another mutation, an in-frame deletion of 9 amino acids resulted in a third membrane spanning domain (ΔI500-F508).
Disintegrate. Two missense mutations also affect the membrane spanning domain, A561V in the S5 domain and N470D in S2. Both mutations affect amino acids conserved in the eag family of potassium channels and appear to alter the secondary structure of the protein. Novel mutation, G628
S, occurs in the pore-forming domain. This domain is highly conserved in all potassium channel α-subunits. This mutation affects a conserved amino acid known to be important for ion selectivity. When this substitution was introduced into Shaker H4, potassium ion selectivity was lost [Hagi
nbotham et al., 1994]. As discussed above, these mutations can cause a loss of HERG function.

The data has implications for the mechanism of arrhythmias in LQT. LQ
Two hypotheses for T have been previously proposed [Schwartz et al., 1994]. Some suggest that dominance of left autonomic innervation causes abnormal cardiac repolarization and arrhythmias. This hypothesis is supported by the finding that removal of the right stellate ganglion can induce arrhythmias in dogs. Furthermore, mythical evidence suggests that some LQT patients are effectively treated by β-adrenergic blocking agents and by left stellate ganglion resection [Schwartz et al., 1994]. LQ
A second hypothesis for T-related arrhythmias suggests that mutations in the cardiac-specific ion channel gene or the gene that regulates cardiac ion channels cause delayed myocyte repolarization. Delayed myocyte repolarization can promote reactivation of L-type calcium channels and can result in secondary depolarization [January and Riddle, 1989]. These secondary depolarizations are the likely cellular mechanisms of Torsades de Pointes arrhythmias [Surawicz, 1989]. This hypothesis is supported by the observation that pharmaceutical blocking of potassium channels can induce QT prolongation and repolarization-related arrhythmias in human and animal models [Anzelevitch and Sicouri, 1994]. LQ
The finding that one form of T is due to a mutation in the cardiac potassium channel gene supports the myocyte hypothesis.

The presence of a cyclic nucleotide binding domain in HERG suggests a mechanism for a link between altered autonomic activity and arrhythmias in LQT. β
-Adrenergic receptor activation increases intracellular cAMP and improves L-type Ca2 ⁺ channel function. Cyclic AMP can also activate HERG, thereby increasing net external currents and accelerating the rate of myocyte repolarization. HERG
Dominant-negative mutations of cAMP would prevent normal regulation of HERG function by cAMP, thereby allowing a dominant effect on L-type Ca2 ⁺ channel function. Imbalance as a result, sympathetic tone, which is facilitated (sympathetic tone) is Ca ^{2 +} channel - to increase the likelihood that may induce dependence secondary passive, promising cellular mechanisms of torsade de Poantsu Will. β-adrenergic blocking agents can act by blocking the effects of cAMP on L-type Ca ²⁺ channels, possibly explaining the beneficial effects of β-blockers in some LQT patients.

This study will have important clinical implications. In recent years, predictive diagnosis has become possible in large families using linkage analysis. Most cases of LQT are sporadic, so genetic testing using linkage analysis cannot be performed. Successive mutation analysis would facilitate genetic testing for LQT. Identification and characterization of genes corresponding to other forms of LQT are necessary for the development of generalized diagnostic tests. Improved diagnostic capacity will allow for rational diagnosis. For example, chromosome 7-linked LQT patients will respond to potassium channel activators such as pinacidil.

Example 12 Generation of Polyclonal Antibodies to HERG A segment of the HERG coding sequence is and expressed as a fusion protein in E. coli. The overexpressed protein is purified by gel elution and used to
Rabbits and mice are immunized using a procedure similar to that described by [Harlow and Lane, 1998]. This procedure is based on Abs for various other proteins.
(See, eg, [Kraemer et al., 1993]).

Briefly, a stretch of the HERG coding sequence was transferred to plasmid PET5A (Novage
n, Inc., Modison, WI) as a fusion protein. After induction with IPTG, overexpression of the fusion protein with the expected molecular weight was determined by SDS / PAGE.
Confirm by The fusion protein is purified from the gel by electroelution. The identity of the protein as a HERG fusion product is confirmed by protein sequencing at the N-terminus. Next, the purified protein is used as an immunogen in rabbits. Rabbit in complete Freund's adjuvant
Immunize with 00 μg of the protein, first add 10 μg in complete Freund's adjuvant.
Boost twice with 0 μg immunogen followed by 100 μg immunogen in PBS at 3 week intervals. Antibodies containing plasma are collected over the next two weeks.

This procedure is repeated to generate antibodies against the mutant form of the HERG gene. These antibodies are used in combination with wild-type HERG to detect the presence and relative levels of mutated forms in various tissues and body fluids.

Example 13 Generation of Monoclonal Antibodies Specific for HERG Monoclonal antibodies are generated according to the following protocol. HE mice intact
RG or, as is well known, HERG peptide conjugated to keyhole limpet hemocyanin with glutaraldehyde or EDC (
(Wild-type or mutant).

The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10-100 μg of the immunogen, and after the fourth injection, a blood sample is taken from the mice to determine if the plasma contains antibodies to the immunogen. Plasma titer E
Quantify by LISA or RIA. Mice with plasma indicating the presence of antigen for the immunogen are selected for hybridoma production.

The spleen is removed from the immunized mouse and a single cell suspension is prepared ([Harlow and Lan
e, 1988]). Cell fusion is performed essentially as described by [Kohler and Milstein, 1975]. Briefly, P3.65.3 myeloma cells (American
(Type Culture Collection, Rockville, MD) is fused with immunized spleen cells using polyethylene glycol as described by [Harlow and Lane, 1988]. Cells are plated at a density of 2 × 10 ⁵ cells / well in a 96-well tissue culture plate. Individual wells were examined for growth and the supernatants of the grown wells were purified by ELISA or RIA with wild-type or mutant HERG target protein.
Check for the presence of ERG-specific antibodies. Cells in positive wells are expanded and subcloned to demonstrate and confirm monoclonality.

Clones with the desired specificity are expanded and grown as ascites in mice or in hollow fiber systems to produce sufficient antibodies for characterization and assay development.

Example 14 Sandwich Assay for HERG A monoclonal antibody is attached to a solid surface, such as a plate, tube, bead, or particle. Preferably, the antibody is attached to the well surface of a 96-well ELISA plate. A 100 μl sample (eg, plasma, urine, tissue cytosol) containing the HERG peptide / protein (wild type or mutant) is added to the solid phase antibody. The sample is incubated at room temperature for 2 hours. Next, the sample liquid is decanted and the solid phase is washed with a buffer to remove unbound material. 1
Add 00 μl of a second monoclonal antibody (for different determinants for the HERG peptide / protein) to the solid phase. The antibody is labeled with a detector molecule (eg, ¹²⁵ I, enzyme, fluorophore, or chromophore) and the second
Is incubated at room temperature for 2 hours. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.

The amount of bound label is proportional to and quantifies the amount of HERG peptide / protein present in the sample. Separate assays are performed using a monoclonal antibody specific for the wild type HERG as well as a monoclonal antibody specific for each mutation identified in HERG.

Example 15 Construction of HERG Expression Plasmid and Transcription of cRNA To facilitate HERG expression in Xenopus oocytes,
The RG cDNA is subcloned into a poly-A ⁺ expression vector and the 5 'and 3' UTRs are reduced to a minimum length. The final HERG expression construct was pSP6
4 Contains the cDNA sequence from nucleotides -6 to 3513 in a plasmid vector (Promega). Prior to use in expression experiments, the HERG construct is characterized by restriction mapping and DNA sequence analysis. Complementary RNA injected into the oocytes was treated with mCAP RNA Capp after linearization of the expression construct with EcoRI.
Prepare with ing Kit (Stratagene).

Example 16 Oocyte Isolation and RNA Injection Xenopus laevis was anesthetized by soaking in 0.2% tricaine for 15-30 minutes. Ovarian lobes were digested with 2 mg / ml type 1A collagenase (Sigma) in Ca ²⁺ -free ND96 solution for 1.5 hours to remove follicular cells. Stages IV and V
Oocytes [Dumont, 1972] were converted to HERG cRNA (0.05 mg / ml, 50 n).
1), and then cultured at 18 ° C. in Bath solution supplemented with 50 μg / ml gentamicin and 1 mM pyruvate. Bath solution (in mM): 88
NaCl, 1 KCl, 0.4 CaCl ₂ , 0.33 Ca (NO ₃ ) 2, 1
_{MgSO 4, 2.4 NaHCO 3, 10} HEPES; containing pH 7.4.

Example 17 Oocyte 2-Microelectrode Voltage Clamp Unless otherwise noted, oocytes were bathed in ND96 solution. This solution (in mM): 9
6 NaCl, 2 KCl, 1 MgCl ₂ , 1.8 CaCl ₂ , 5 HEPES
Containing pH 7.6. In some experiments, KCl was changed by equimolar substitution of NaCl. Currents were recorded at room temperature (21-23 ° C.) using a standard two-microelectrode voltage clamp technique. Fill the glass microelectrodes with 3M KCl and break their tips to break down the voltage-1-3 MΩ and current-to the recording electrode.
A tip resistance of 0.6 to 1 MΩ was obtained for the passing electrode. The oocytes were transferred to Dagan T
The voltage was clamped with an EV-200 amplifier. Voltage instructions were converted to pClamp software (ver. 6, Axon Instruments), 486DX2 personal computer and T
Generated using the L-1 D / A interface (Axon Instruments). The current signal is converted to a low-frequency cutoff frequency (−3 db) of the quadrupole Bessel filter of 2 to 4
Digitally sampled at a rate equal to twice. Unless otherwise noted, currents were corrected for leak and capacitance using a standard online P / 3 leak subtraction. Oocyte membrane potential was kept at -70 mV between test pulses applied at a rate of 1-3 pulses / min. Data analysis included exponential fitting of current trajectories, pCLAM
Performed with P. Fitting the appropriate data to the Boltzmann function or Goldman-Hodgkin-Katz constant field equation was performed using Kaleidagraph (Synergy Software). Data are expressed as mean ± SEM (n = number of oocytes).

Although the invention has been disclosed in this patent application by reference to details of preferred embodiments of the invention, it is to be understood that the disclosure is intended to be illustrative rather than limiting. At the same time, it is contemplated that modifications will readily occur to those skilled in the art in the spirit of the invention and the appended claims.

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[0236]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the current induced by the step of depolarizing a potential difference in Xenopus oocytes injected with HERG cDNA.

FIG. 2 shows the kinetics of HERG current activation and deactivation.

FIG. 3 shows that the reverse potential of the HERG current has changed in [K ⁺ ] _e as expected for the K ⁺ -selective channel.

FIG. 4 is a diagram showing activation of HERG current by extracellular K ⁺ .

FIG. 5 shows HERG rectification resulting from rapid inactivation.

FIG. 6 is a diagram showing that the HERG current is blocked by La ³⁺ .

FIG. 7 shows a physical map and exon organization of HERG.

FIG. 8 shows the genomic organization of the HERG coding sequence and the 5 ′ and 3 ′ untranslated sequences.

FIG. 9 shows pedigree and genotype analysis of 5 new LQT families.

FIG. 10 shows deletions in the HERG gene associated with LQT in two families.

FIG. 11 shows a blood related structure of K2595, SSCP using primer pair 1-9.
FIG. 2 shows the results of the analysis and shows that abnormal SSCP conformers co-segregate with disease in this family.

FIG. 12 shows HERG point mutations identified in three LQT relatives.

FIG. 13 shows a HERG missense mutation associated with LQT.

FIG. 14 shows the HERG de novo mutation in sporadic cases of LQT.

FIG. 15 shows Northern blot analysis of HERG mRNA showing strong expression in heart.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 1/21 4H045 1/21 C12Q 1/02 5/10 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/566 33/577 B 33/566 C12P 21/08 33/577 C12N 15/00 ZNAA / / C12P 21/08 5/00 AB (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW) , SD, SL, SZ, UG, ZW), E (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Igoa Sproutski 02134 Chester Street 97, Allston, Mass., Apartment A-2 F Term (reference) 2G045 AA35 BA14 BB14 BB24 CB01 DA13 DA36 FA20 FB02 FB03 FB05 FB12 4B024 AA01 AA11 BA53 CA01 CA04 CA09 DA01 DA02 DA06 DA11 DA12 EA04 GA11 HA12 HA15 HA17 4B063 QA01 QA05 QA18 QA19 QQ03 QQ08 QQ43 QQ89 QR14 QR32 QRSQ QRQ QRQ QRQ QRQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQYQYQYQQQQQQQQQRQBQ either DA13 4B065 AA19X AA26X AA41X AA58X AA72X AA88X AA90X AA92X AA93Y AB01 AB05 AC14 BA02 BA08 CA24 CA25 CA44 CA46 4H045 AA10 AA11 AA30 BA10 CA40 DA75 DA76 EA23 EA50 FA72 FA73 FA74 HA07

Claims (30)

[Claims] 1. An isolated DNA comprising a nucleic acid of SEQ ID NO: 1 containing a change, wherein the change is T at position 1714, G at position 1762, T at position 1841, C at position 1955, and a table. 7. An isolated DNA, which is selected from the group consisting of all the mutations listed in 7. 2. The nucleic acid probe of claim 1, which hybridizes to the isolated DNA under conditions that will not hybridize to the nucleic acid of SEQ ID NO: 1. 3. The method according to claim 2, comprising hybridizing the probe according to claim 2 to a DNA or RNA patient sample, wherein the presence of a hybridization signal is an indicator of the long-term QT syndrome. A method for diagnosing the mutations that cause it. 4. The DNA or RNA of a patient is amplified, and the amplified DNA is amplified.
Alternatively, the method according to claim 3, wherein the RNA is hybridized with the probe according to claim 2. 5. The method according to claim 3, wherein the hybridization is performed in situ. 6. The method of claim 3, wherein said assay is performed using nucleic acid microchip technology. 7. A method for diagnosing a mutation causing long-term QT syndrome, comprising using a single-stranded DNA conformation polymorphism analysis technique, wherein the method comprises: 1) SEQ ID NO: 56 and 57; 2) SEQ ID NOs: 58 and 59; 3) SEQ ID NOs: 60 and 61; 4) SEQ ID NOs: 62 and 63; 5) SEQ ID NOs: 64 and 65; 6) SEQ ID NOs: 66 and 67; 8) SEQ ID NOs: 70 and 71; 9) SEQ ID NOs: 72 and 73; 10) SEQ ID NOs: 74 and 75; 11) SEQ ID NOs: 76 and 77; 12) SEQ ID NOs: 78 and 79 13) SEQ ID NOs: 80 and 81; 14) SEQ ID NOs: 82 and 83; 15) SEQ ID NOs: 84 and 85; 16) SEQ ID NOs: 86 and 87; 17) SEQ ID NOs: 88 and 9; 18) SEQ ID NO: 90 and 91; 19) SEQ ID NO: 92 and 93; and 20) SEQ ID NO: 94 and 95; wherein the use of primer pairs selected from the group consisting of. 8. A method for diagnosing a mutation causing long-term QT syndrome, comprising amplifying a region of the HERG gene or RNA and then sequencing the amplified gene or RNA, wherein the method comprises the steps of: Is the following group: SEQ ID NO:
1 at base 1714, G at base 1762 of SEQ ID NO: 1, T at base 1841 of SEQ ID NO: 1, C at base 1889 of SEQ ID NO: 1 and the mutations shown in Table 7,
Or a mutation indicated by any one or more of the following: 9. A method of diagnosing a mutation causing long-term QT syndrome, comprising identifying a mismatch between a patient's DNA or RNA and a wild-type DNA or RNA probe, wherein the probe comprises Hybridizes to a DNA or RNA region, wherein the region comprises the following groups: T at base 1714 of SEQ ID NO: 1, G at base 1762 of SEQ ID NO: 1, T at base 1841 of SEQ ID NO: 1, A method comprising any one of C at base 1889 of SEQ ID NO: 1 and a region containing the mutation shown in Table 7. 10. The method of claim 9, wherein said mismatch is identified by an RNase assay. 11. A mutant HERG polypeptide that binds to a wild-type HE
An antibody that does not bind to an RG polypeptide, wherein said mutant polypeptide causes long-term QT syndrome, wherein said mutant polypeptide is the polypeptide of SEQ ID NO: 2, wherein said polypeptide is : Cysteine at amino acid residue 572;
An antibody having a mutation selected from the group consisting of aspartic acid at amino acid residue 588, valine at amino acid residue 614, alanine at amino acid residue 630 and the mutations shown in Table 7. 12. The antibody according to claim 11, wherein said antibody is a monoclonal antibody. 13. A method for diagnosing long-term QT syndrome, comprising assaying for the presence of a mutated HERG polypeptide in a patient by reacting a patient sample with the antibody of claim 11. A method wherein the presence is an indicator of long-term QT syndrome. 14. The method according to claim 13, wherein said antibody is a monoclonal antibody. 15. The method according to claim 13, wherein said assay comprises immunoblotting. 16. The method according to claim 13, wherein said assay comprises an immunohistochemical technique. 17. An isolated HE containing a mutation causing long-term QT syndrome
RG polypeptide wherein the mutation is a cysteine at amino acid residue 572, an aspartic acid at amino acid residue 588, a valine at amino acid residue 614, an alanine at amino acid residue 630, or a mutation shown in Table 7. An isolated HERG polypeptide, characterized in that: 18. A method for diagnosing long-term QT syndrome comprising a HERG polypeptide, wherein a mutation in said polypeptide is an indicator of long-term QT syndrome, wherein said mutation comprises amino acid residue 572 Cysteine, amino acid residue 588
Aspartic acid, valine at amino acid residue 614, alanine at amino acid residue 630 and the mutations shown in Table 7. 19) 1) SEQ ID NOs: 56 and 57; 2) SEQ ID NOs: 58 and 59; 3) SEQ ID NOs: 60 and 61; 4) SEQ ID NOs: 62 and 63; 5) SEQ ID NOs: 64 and 65; 6) SEQ ID NOs: 66 and 67; 7) SEQ ID NOs: 68 and 69; 8) SEQ ID NOs: 70 and 71; 9) SEQ ID NOs: 72 and 73; 10) SEQ ID NOs: 74 and 75; 11) SEQ ID NOs: 12) SEQ ID NOs: 78 and 79; 13) SEQ ID NOs: 80 and 81; 14) SEQ ID NOs: 82 and 83; 15) SEQ ID NOs: 84 and 85; 16) SEQ ID NOs: 86 and 87; 18) SEQ ID NOs: 90 and 91; 19) SEQ ID NOs: 92 and 93; and 20) SEQ ID NOs: 94 and 95 A pair of nucleic acid to be selected. 20. A method for amplifying an exon of HERG, comprising using a pair of primers selected from the primer pair according to claim 19. 21. An isolated nucleic acid comprising SEQ ID NO: 3. 22. a) A first set of cells expressing HERG with a mutation is placed in an immersion solution to measure a first induced K ⁺ current, wherein the mutation is a cysteine at amino acid residue 572. , Aspartic acid at amino acid residue 588, valine at amino acid residue 614, alanine at amino acid residue 630, or a mutation shown in Table 7; b) measuring the first induced K ⁺ current; c) wild Placing a second set of cells expressing type HERG in the immersion solution and measuring a second induced K ⁺ current; d) measuring the second induced K ⁺ current; e) the immersion solution of step (a) F) measuring the third induced K ⁺ current of the cell in step (e); then g) the third induced K ⁺ current is more second than the first induced K ⁺ current to determine whether similar by induced K ⁺ current A method for screening agents which are useful in treating individuals with mutations in non HERG, wherein the said mutation, cysteine amino acid residues 572, aspartic acid at amino acid residue 588,
A valine at amino acid residue 614, an alanine at amino acid residue 630, or a mutation shown in Table 7, wherein the third is closer to the second induced K ⁺ current than the first induced K ⁺ current. A screening method, characterized in that an agent that produces an induced K ⁺ current is useful for treating the individual. 23. The cells of the first set of cells are transfected with a mutant HERG, wherein the mutant HERG comprises a cysteine at amino acid residue 572, an aspartic acid at amino acid residue 588, and a valine at amino acid residue 614. 23. The method of claim 22, which encodes a HERG protein having alanine at amino acid residue 630 or a mutation shown in Table 7. 24. The method of claim 22, wherein the cells of the second set of cells are transfected with a nucleic acid encoding wild-type HERG. 25. The method of claim 22, wherein said first set of cells or said second set of cells is obtained from a transgenic animal. 26. A vector comprising the isolated DNA according to claim 1. 27. A cell transfected with the vector according to claim 26. 28. A cell transfected with the DNA of claim 1. 29. A non-human transgenic animal comprising the DNA according to claim 1. 30. A non-human transgenic animal comprising the vector according to claim 26.